Chapter 2

Review of Literature

Cancer is a worldwide public health problem. Despite considerable progress in its

early diagnosis and treatment, successful remedy is alarmingly negligible. Cancer was

thought to arise when cell growth exceeds the rate at which cells die, so that cells are

dividing at an uncontrollable rate. There are more than 100 different types of cancer.

Most cancers are named for the organ or type of cell in which they start. Cancer types can

be grouped into three broader categories. The main categories of cancer include:

Carcinoma - (cancer that begins in the skin or in tissues that line or cover internal

organs), Sarcoma - (cancer that begins in bone, cartilage, muscle, blood vessels, or other

connective or supportive tissue), Leukemia - (cancer that starts in blood-forming tissue

such as the bone marrow and causes production of large numbers of abnormal blood cells

and enter the blood).

In current anti-cancer therapy, drugs are administered though intravenous and oral

route using conventional formulations like tablet, capsule and injectable. Sustained and

targeted delivery of anti-cancer agents at the site of action is desired to maximize the

killing effect during the tumor growth phase and avoiding the exposure to surrounding

healthy cells for reducing the toxicity. It is also desired to maintain a steady state infusion

of drug into the tumor interstitium to maximize the exposure to the dividing cells that

results in tumor regression (Shenoy and Amiji, 2005). Conventional oral and injectable

dosage forms of anti-cancer drugs are not able to do this due to short biological half-life,

narrow therapeutic index, poor oral bioavailability and formulation difficulties like poor

water solubility, stability and high molecular weight (Jain, 1994). In the recent past,

advances in novel drug delivery system (NDDS) have resulted in use of several colloidal

carriers such as liposomes, niosomes, microemulsion, nanoemulsion, microsphere and

polymeric micelles for sustained and targeted delivery of anti-cancer agents. Further,

Chapter 2 - Review of literature

-10-

revolutions in nanotechnology increased the hope for rationalization in therapeutic

options for rationalized delivery of anti-cancer agents with high efficacy. Development

of NDDS based formulation for delivery of anti-cancer drugs is a recent topic of research

in Pharmaceutical Industries. Nanoxel(R)

, nanoparticles based formulation for paclitaxel

from Dabur and Abraxane(R)

, albumin based formulation for paclitaxel from Abraxis

BioScience Inc., USA are the well known commercial products (Table 2.1). The reason

behind the interest of pharmaceutical company in this area of research is due to high cost

of treatment, required repeated administration for prolong period of time and exponential

increase in number of cancer patients. In the present section of review of literature, we

have explored the possible use of different carrier systems for sustained and targeted

delivery of anti-cancer agents with their relative advantages, limitations and commercial

importance. This information will helps the drug delivery scientist in designing the better

formulation for delivery of anti-cancer drugs.

Table 2.1: Commercially available NDDSs for anti-cancer drugs.

Drug name Novel system Brand name Company

Paclitaxel Albumin bound particles Abraxane Abraxis Bioscience

LLC

Paclitaxel Polymeric nanoparticles gel Nanoxel Dabur India Ltd.

Doxorubicin Liposomal injection Doxil Ortho Biotech

Doxorubicin PEGylated liposomal

injection

Lip-Dox TTY Biopharm

Doxorubicin Liposomal Injection Caelyx Schering-Plough

Doxorubicin PEGylated liposomal

injection

Myocet Cephalon Inc.

Doxorubicin PEGylated liposomal

injection

Lipo-dox Sun

pharmaceuticals

Cytarabine Liposomal injection Depocyt Enzone

Pharmaceutical

2.1 Pathophysiology of Cancer

Cancer is basically a disease of failure of regulation of cell cycle. In cancer, the

cells transform from normal into cancer cells mainly due to alterations in genes which

regulate the cell growth and differentiation. The altered genes are divided into two broad

categories. Oncogenes (e.g. Her 2, c-Myc, etc.) and tumor suppressor genes p53 Rb).

Oncogenes are the genes which promote the cell growth and reproduction. Second class

of genes inhibits the cell division. The cancerous transformation can occur through the

Chapter 2 - Review of literature

-11-

formation of novel oncogenes, the inappropriate over expression of normal oncogenes or

disabling of tumor suppressor genes. Genetic changes can occur at different levels and by

different mechanisms. The gain or loss of an entire chromosome can occur through errors

in mitosis. Cell division is a genetic process in which a cell passes its genes onto two

daughter cells, each of which is a clone or exact of itself. Sometimes, this orderly process

goes wrong, the genes in a cell may suffer a mutation or some mistakes may occur in

DNA replication and recombination during cell division. Genetic changes are more

commonly by mutations, which are changes in the nucleotide sequence of genomic DNA.

Large-scale mutations involve the deletion or gain of a portion of a

chromosome. Genomic amplification occurs when a cell gains many copies (often 20 or

more) of a small chromosomal locus, usually containing one or more oncogenes and

adjacent genetic material. Translocation occurs when two separate chromosomal regions

become abnormally fused, often at a characteristic location. Small-scale mutations

include point mutations, deletions, and insertions, which may occur in the

promoter region of a gene and affect its expression, or may occur in the gene's coding

sequence and alter the function or stability of its protein product. Disruption of a single

gene may also result from integration of genomic material from a DNA

virus or retrovirus, and resulting in the expression of viral oncogenes in the affected cell

and its descendants. Replication of the enormous amount of data contained within the

DNA of living cells will probabilistically result in some errors (mutations). Complex

error correction and prevention is built into the process, and safeguards the cell against

cancer. If significant error occurs, the damaged cell can "self-destruct" through

programmed cell death, termed apoptosis. If the error control processes fail, then the

mutations will survive and be passed along to daughter cells. Some environments make

errors more likely to arise and propagate. Such environments can include the presence of

disruptive substances called carcinogens, repeated physical injury, heat, ionising

radiation, or hypoxia. The transformation of normal cell into cancer is akin to a chain

reaction caused by initial errors, which compound into more severe errors, each

progressively allowing the cell to escape the controls that limit normal tissue growth.

This rebellion-like scenario becomes an undesirable survival of the fittest, where the

driving forces of evolution work against the body's design and enforcement of order.

Once cancer has begun to develop, this ongoing process, termed clonal evolution drives

progression towards more invasive stages. Figure 2.1 shows pathophysiology of cancer.

Chapter 2 - Review of literature

-12-

Figure 2.1: Pathophysiology of cancer

(www.rnspeak.com/pathophysiology/cancer)

Chapter 2 - Review of literature

-13-

2.2 Problems in Conventional Delivery of Anti-Cancer Drugs

Oncology is undoubtedly the most rapidly growing subspecialty in the field of

medicine. There are so many treatment options for cancer treatment such as surgery,

radiation therapy, immunotherapy, hormonal therapy and chemotherapy. Among these,

chemotherapy is mainly used for cancer treatment, but is associated with considerable

side-effects. Table 2.2 summarizes the classification of anti-cancer drugs. Presently, anti-

cancer agents are administered through conventional oral and intravenous routes. Table

2.3 summarizes the marketed conventional dosage forms for anti-cancer drugs. These

routes have shown significant side effects because non-specific delivery of anti-cancer

drugs to healthy organs. Oral route is mainly preferred in the first place for its

convenience and its potential to improve patient quality of life. In addition, this

administrative route is economical and also eliminates the cost for hospitalization.

Despite of these obvious advantages, this route has many limitations for delivery of anti-

cancer drugs such as short biological half-life, poor patient compliance, low therapeutic

index (TI), development of resistance, inability to achieve therapeutic concentrations at

the target site and insufficient bioavailability due to limited aqueous solubility

(Lowenthal and Eaton, 1996; Klein-Szanto,1992), degradation in gastro-intestinal fluids

and/or affinity for intestinal and liver cytochome P450 (CYP3A4) and P-glycoprotein (P-

gp) (De Mario and Ratain,1998).

The substantial patient variability in bioavailability after oral administration

represents another major limitation. Differences in absorption profile may result in

significant differences in pharmacologic effects. Intravenous route also has many

limitations particularly as this route delivers potentially high concentration of drug to

normal tissues which results in toxicity. Several anti-cancer agents are biologically

reactive and may trigger the release of various vasoactive substances, sometimes

resulting in life threatening reactions. In addition, for intravenous administration, the drug

requires adequate aqueous solubility. Since many anti-cancer agents lack adequate drug

solubility and stability properties, co-solvents and other solubilization techniques are

often required Jonkman de-Vries et al., 1996; Watkin., 1979). Paclitaxel is currently

formulated in vehicle composed of 1:1 blend of Cremophor EL (polyethoxylated castor

oil): ethanol which is diluted 5-20 folds with normal saline or dextrose (5%) for

Chapter 2 - Review of literature

-14-

intravenous administration (Szebeni et al., 1998). This formulation has limited stability

on dilution and is associated with significant vehicle related problems in clinic such as

hyperlipidaemmia and abnormal lipoprotein aggregation of erythocytes (Weiss et al.,

1990). In addition, Cremophor EL also showed various serious side effects such as

hypersensitivity reactions, nephrotoxicity, neurotoxicity, cardiotoxicity (Gelderblom,

2001). Direct injection of a cytotoxic agent into the hepatic artery resulted in increased in

hepatic extraction of selected drugs and consequently gave increased systemic exposure.

Many common solid tumors, including breast, brain and prostate tumors did not respond

well to conventional systemic chemotherapy (Hunter et al., 1997).

2.3 Mechanism of Anti Cancer Drugs

The broad mechanism of cell specific anti-cancer drugs is that they can act during

a specific phase of the cell cycle. Figure 2.2 shows the specific mechanism of action of

different anti-cancer drugs.

Figure 2.2: Effect of anti cancer drugs on cell cycle.

Chapter 2 - Review of literature

-15-

Table 2.2: Classification of anti-cancer drugs.

S. No. Class of drugs Examples Therapeutic

indications

Side effects Mechanism of action

1. Alkylating

Agents Ifosamide

Cyclophosphamide

Busulfan

Cholrambucil

Carmustine

Decarbazine

Testicular, bladder,

neck, hodgkins,

chronic lymphatic

leukaemia

Bone marrow

suppression

Leukopenia

Hemorrhage

Thrmbocytopenia

Alkylation of target DNA

Crosslinkage of the DNA

Fragmentation of DNA

2. Anti-

Metabolites Marcaptopurine

Thioguanine

Fludarabine

Fluorouracil

Cytarabine

Methotrexate

Acute leukemia,

Choriocarcinoma,

non Hodgkin’s

lymphoma, colon,

urinary baldder, liver

and breast cancers

Chills, fever,

vomiting after

injection and

opportunistic

infections

Inhibition of thymidilate

synthase

Inhibition of DNA

polymerase

Inhibition of nucleotide

metabolism

3. Antibiotics Dactinomycin

Danuorubicin

Doxorubicin

Idarubicin

Valrubicin

Bleomycin

Mitomycin

Plicamycin

Whilm’s tumor,

Rhabdomyosarcoma,

solid tumors, acute

leukaemia, acute non

haemolytic

leukaemia, non

Hodgkin’s

lymphoma, skin

cancer

Mucosal

inflammation,

pulmonary fibrosis

Inhibit DNA and RNA

synthesis

4. Natural

Products Vincristine,vinblastine

Taxanes:Paclitaxel,

Docetaxel

Etoposide

Taniposide

Campothecins

Docetaxel

Solid tumors,

childhood acute

leukaemia, Wilms

tumor, Lung

carcinaoma

Reversible

myelosuppression

mucositis,

neurotoxicity,

peripheral

neuropathy and

alopecia.

Disrupt microtubule

apparatus

Inhibit depolymeriyation

Chapter 2 - Review of literature

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5. Miscellaneous Cisplatin

Carboplatin

Hydroxyureas

Asparaginase

Mitoxantrone (MTO)

Gallium Nitrate

Arsenic trioxide

Bexarotene

Filgrastim

Chronic myeloid

leukaemia, oat cell

carcinoma, non

Hodgkin’s

lymphoma,

Myelosupression,

testicular and

ovarian carcinoma

Leucopenia,

Thrombocytopeni,

Myelosupression,

Vomiting

Cross links DNA

chromosomal breaks

Reduce level of L-

asparagine

6. Hormones Tamoxifen

Prednisone

Dexamethasone

Progesteron

Testosterone propionate

Flutamide ,

GnRH Analogue:

Leuprolide, Buserelin

Growth Hormone,

glucagon and insulin

inhibitor: Octreotide

Modify the growth

of hormone

dependent tumors,

Acute childhood

leukemia, prostate

carcinoma

Tumor flare pain,

breast tenderness,

memory problems,

weight gain,

depression and

osteroprosis.

The exact mechanisms of

action of hormones are

not clear and may involve

in both direct effect on the

tumor cells and indirect

endocrine effects.

Chapter 2 - Review of literature

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Table 2.3: Marketed conventional dosage forms of anti-cancer drugs.

S. No. Drug Dosage Form Brand Company

1. Chlorambucil Film coated

tablet

Amcil Amronco life

sciences limited

2. Methotrexate Tablet Hitrex VHB Life sciences

Limited

3. Tamoxifen Tablet Nolvadex AstraZeneca

Pharmaceuticals

4. Busulfan Tablet Busulfex Orphan drug company

5. 5-FU Capsules Fluracil Biochem

Pharmacuticals

6. Docetaxel Injection Taxotere Sanofi Aventis

7. Cisplatin Injection Amcis Amronco life

sciences limited

8. Docetaxel Injection Neudoc Claris

9. 5-FU Injection Adrucil Teva parentral

Medicines

11. Vincristine Injection Oncovin Genus

Pharmaceuticals

Limited

12. Cyclophosphamide Injection Cycrame VHB Life sciences

Limited

13. Bleomycin Injection Blenoxane Brisol-Myers Squibb

Company

14. Paclitaxel Injection Taxol Brisol-Myers Squibb

Company

15. Methotrexate Injection Trexall Duramed

Pharmaceuticals, Inc

16. Doxorubicin HCl Injection Adriamycin

RDF

Pharmacia Inc.

17. 5-FU Topical Cream Efudex Valeant

Pharmaceuticals. Inc

2.4 Novel Drug Delivery Systems for Anti-Cancer Drug Delivery

Currently, with conventional cancer chemotherapy, cytotoxic agent’s travel

throughout the body delivering powerful medication to all the cells they encounter, both

healthy and cancerous. When healthy cells are damaged by unnecessary medication, a

patient can experience unpleasant side effects ranging from hair loss to nausea.

Conventional cancer chemotherapy also damages the patient’s immune system. The

pharmaceutical scientists continue to find innovative ways to treat cancer. Drug delivery

scientists have the task of making that anti-cancer drugs should reach the target site in the

Chapter 2 - Review of literature

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body in the required quantities and at the right time. The new innovations in delivering

cancer therapies are the development of a technology platform which targets the therapy

only to the tumor; leaving normal cells undamaged (Table 2.4). The impetus for the

development of NDDSs apart from therapeutic efficacy, are the cost. The development

cost of a new drug may be about $250-300 million and takes about 12-15 years to reach

the market place, whereas an existing drug molecule can get a second life with NDDS

that can be developed in half the time and at 20% of the cost of a new drug discovery.

The cost per milligram of drug delivered though NDDS is more expensive than

conventional peroral administration and can only be justified if NDDS improves

therapeutic efficacy, patient compliance and reduces toxic side effects. NDDSs deliver

drugs directly to its target tissue and moreover with this approach much higher local drug

concentrations can be achieved compared to traditional approaches. Figure 2.3 shows the

mechanism for sustained and targeted delivery of anti-cancer agents.

Figure 2.3: Mechanism for sustained and targeted delivery of anti-cancer agents using NDDS

Chapter 2 - Review of literature

-19-

2.5 Different Approaches Reported for Sustained and Targeted Delivery

of Anti-Cancer Agents

2.5.1 Nanoparticles

The development of nanoparticles for drug delivery began in the 1960s (Kreuter,

2007). Nanoparticles (NPs), as the name implies, are particles varying in size from 10 to

1000 nm and contain drug in encapsulated or absorbed form. The drug may be attached to

a nano particle matrix, or dissolved, encapsulated and entrapped, giving rise to different

terminologies as nanoparticles, nanospheres or nanocapsules. All these terms signify their

most general characteristic, i.e. they are nano sized particles. Anti-cancer agent loaded

nanoparticles constitute an almost versatile drug delivery system, with their ability to

overcome physiological barriers and guide the drug to specific cells or intracellular

compartments either by passive or ligand-mediated targeting mechanisms (Pinto Reis et

al.,2006; Hamidi et al.,2008; Sahoo and Labhasetwar, 2003; Vasir et al.,2005).

Nanoparticles based drug delivery systems have many advantages for anti-cancer

drug delivery such as pass through the smallest capillary vessels because of their ultra-

tiny volume and avoid rapid clearance by phagocytes so that their duration in blood

stream is greatly prolonged (Jung et al., 2000) (Table 2.5). Nanoparticles can also

penetrate cells and tissue gap to arrive at target organs such as liver, spleen, lung, spinal

cord and lymph. They could show controlled release properties due to the

biodegradability, pH, ion and/or temperature sensibility of materials. All these properties

can improve the utility of anti-cancer drugs and reduces the toxic side effects. Following

section summarized the salient findings of use of nanoparticles for delivery of anti-cancer

agents.

Paclitaxel is one of the best found anti-cancer drug and current commercial

formulation employed Cremophor EL as adjuvant for its solubilization. Mu and Feng,

(2003) proposed d-α tocopheryl polyethylene glycol 1000 succinate (vitamin E TPGS) a

novel surfactant as well as matrix material with other biodegradable polymers for

fabrication of nanoparticle formulation of paclitaxel. Obtained results indicated that

vitamin E TPGS could be an efficient emulsifier for fabrication of polymeric

nanoparticles by the single emulsion technique. TPGS may also have the potential to

improve nanoparticle adhesion to cells and the hemodynamic properties of the nano

particles in the blood flow. Finally, it was concluded that vitamin E TPGS is

Chapter 2 - Review of literature

-20-

advantageous either as emulsifier or as matrix material blended with (PLGA) for the

manufacture of nanoparticles for controlled release of paclitaxel. In another study, Feng

et al. (2002) have developed paclitaxel loaded nanospheres formulation to achieve better

therapeutic effects with minimum side effects. In this investigation phospholipids,

cholesterol and vitamins were used to replace traditional chemical emulsifiers to achieve

high encapsulation efficiency (EE) and desired release rate of the drug. The in vitro

release measurement showed that the release of paclitaxel could last more than 3 months

at an approximately constant release rate after an initial burst. Finally, drug delivery

scientists concluded that phospholipids as well as other natural emulsifiers such as

cholesterol and vitamins may have great advantages for preparation of polymeric

nanospheres for controlled release of paclitaxel as well as other anti-cancer drugs. PEG-

coated biodegradable polycyanoacrylate nanoparticle (PEG-nanoparticles) conjugated to

transferrin for sustained and targeted delivery of paclitaxel was studied by Xua et al.

(2005). They have found that sustained release profile of paclitaxel from developed

nanoparticle formulation and release was sustained over 30 days (81.6%) period of time.

Nanoparticle formulation was also found to exhibit a markedly delayed blood clearance

in mice, and the paclitaxel level from conjugated nanoparticles remained much higher at

24 h compared with that of free drug from paclitaxel injection. The biodistribution

profiles of nanoparticles in S-180 solid tumor bearing mice after intravenous

administration showed the tumor accumulation of paclitaxel increased with time and the

paclitaxel concentration in tumor was found to be 4.8 and 2.1 times higher than those

from paclitaxel injection and PEG-nanoparticles at 6 h after intravenous injection.

Authors hypothesized that PEG-coated biodegradable polycyanoacrylate nanoparticle

conjugated to transferrin could be an effective carrier for paclitaxel delivery. In further

study, Zhang and Feng (2007) reported the use of poly(lactide)-tocopheryl polyethylene

glycol succinate (PLA-TPGS) as novel synthesized copolymers having desired

hydrophobic-hydrophilic balance for delivery of paclitaxel. Nanoparticle formulation of

paclitaxel using PLA-TPGS copolymer was prepared and characterized in vitro and ex

vivo. Authors have compared the cellular uptake and anti-cancer activity of developed

nanoparticle formulation with commercial paclitaxel injectable formulation (Taxol(R)

)

using HT-29 and Caco-2 cells. The results indicated significantly higher anti-cancer

activity and reduced cytotoxicity as measured by (MTT) assay of nanoparticle

formulation of paclitaxel.

Chapter 2 - Review of literature

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Table 2.4: Summary of findings reported for altering the biodistribution of anti-cancer drugs.

Drug Name System Finding References

Paclitaxel Nano

particles Achieved larger cytotoxicity and

smaller IC50 over Commercial

preparation

Zhang and

Feng,(2007)

Mitoxantrone

(MTO)

Nanospheres Found as promising carrier with

altered biodistribution

Lu et

al.,(2006)

Tamoxifen Nanoparticles Increased level of accumulation of the

drug within tumor

Shenoy and

Amiji,

(2005)

Doxorubicin Human albumin

serum (HAS)-

Nanoparticles

Diminish the toxicity and overcome

the problem of multi drug resistance

Dreis et al.

(2007)

Paclitaxel Microemulsion Enhanced anti-tumor activity

Total inhibition of cell growth upto

144h

Kang et

al.,(2004)

Paclitaxel O/W emulsion Promising carrier for paclitaxel

Average life span of ascetic tumor

bearing mice was prolonged

Kan et

al.,(1999)

Docetaxel Water Emulsion

system Enhanced accumulation of docetaxel

in a model tumor

4.5 fold increase accumulation of

docetaxel in model tumor mice

Yanasarn et

al.,(2009)

9-nitro-

camptothecin

(9-NC)

Folate-conjugated

polymer micelles 3.7 to 17.0 times increased killing

ability shown by formulated

preparation than free drug in various

cell lines

Hana et

al.,(2009)

Docetaxel Solid lipid

Nanoparticles

(SLNs)

Low systemic toxicity Zhenghong

et al.,(2009)

5-Fluorouracil

(5-FU)

Niosomes 4 to 8 folds enhanced drug penetration Cosco et

al.,(2009)

Methotreaxate Liposomal

formulation 43 folds decreased in resistance of

tumor cells to methotrexate

Vodovozov

a et

al.,(2007)

Doxorubicin Liposomes Enhanced anti-tumor activity Pakunlu et

al.,(2006)

Mitoxantrone PEGylated

liposomes Optimized formulation enhanced

6459 fold AUC and also increases

anti-tumor activity in comparison to

drug solution

Chun Lei et

al.,(2008)

SN-38 Liposomes 200 to 2000 fold more cytotoxicity

than free drug

Zhang et

al.(,2004)

Chapter 2 - Review of literature

-22-

Shenoy and Amiji, (2005) evaluated and compared the biodistribution profile of

tamoxifen administered intravenously (i.v.) as a simple solution and encapsulated in

polymeric nanoparticulate formulations, with or without surface-stabilizing agents. Poly

(ethylene oxide)-modified poly (ethylene oxide-caprolactone) (PEO-PCL) nanoparticles

with an average diameter of 150-250 nm, having a smooth spherical shape, and a positive

surface charge were obtained with the formulation procedure. About 90% drug

encapsulation efficiency was found when tamoxifen was loaded at 10% by weight of the

polymer. The primary site of accumulation for the drug-loaded nanoparticles after i.v.

administration was the liver, though up to 26% of the total activity was recovered in

tumor at 6 h post-injection for PEO-modified nanoparticles. PEO-PCL nanoparticles

exhibited significant increase in tumor localization as well as extended their presence in

the systemic circulation than the controls (unmodified nanoparticles or the solution form).

Lu et al. (2006) have evaluated tissue distribution, acute toxicity and therapeutic

efficiency against breast cancer and its lymph node metastases of formulated bovine

serum albumin (BSA) and chitosan (CS) nanospheres of mitoxantrone (MTO). After

local injection in rats, MTO nanospheres (NS) showed a slower elimination rate and a

much higher drug concentration in lymph nodes compared with MTO solution, and a

lower drug concentration in other tissues. There was no observed acute toxicity to the

main tissues of Kunming mice after local injection of MTO-BSA-NS. The inhibition rate

of the nanospheres against breast cancer was much higher than that of MTO solution, and

lymph node metastases were efficiently inhibited by the nanospheres, especially MTO-

BSA-NS. The results showed that nanospheres seem to be a promising carrier system for

delivery of anti-tumor agents to breast cancer and especially for its lymph node

metastases.

Dries et al. (2007) studied human albumin serum (HAS) nanoparticles for

delivery of doxorubicin. The influence on cell viability of the resulting nanoparticles was

investigated in two different cell lines UKF-NB-3 and IMR-32. The anti-cancer effect of

the drug-loaded nanoparticles was found to increase significantly in comparison to

doxorubicin solution. Authors concluded that HSA nanoparticles represent promising

Chapter 2 - Review of literature

-23-

drug carrier systems for anti-cancer drug delivery and may diminish their toxicity,

optimize body distribution and overcome multi drug resistance.

Sun et al. (2008) studied nanoparticle formulation containing polymer poly D,L-

lactide-co-glycolide/montmorillonite (PLGA/MMT) with human epidermal growth

factor receptor-2 (HER-2) antibody Trastuzumab for targeted breast cancer chemotherapy

with paclitaxel as a model anti-cancer drug. The results of in vitro drug release study

found that nanoparticle formulation exhibited a biphasic drug release with a moderate

initial burst release followed by sustained release profile. Surface chemistry analysis was

conducted by X-ray photoelectron spectroscopy, which confirmed the presence of

Trastuzumab on the nanoparticles surface. The results of in vitro cytotoxicity experiment

on SK-BR-3 cells further proved the targeting effects of the antibody decoration judged

by IC50 after 24 h culture, the therapeutic effects of the drug formulated in the

nanoparticles with surface decoration was found 12.74 times higher than that of the bare

nanoparticles and 13.11 times higher than commercial paclitaxel formulation (Taxol)®.

Chakravarthi et al. (2010) compared the anti-tumor efficacy of paclitaxel loaded

nano particles and delivered intratumorally in comparison to marketed Cremophor EL

based paclitaxel injection. It was found that developed nanoparticles sustained the drug

release, increased cellular concentration and enhanced anti-tumor efficacy of paclitaxel

compared to marketed formulation.

Wang et al. (2011a) prepared paclitaxel loaded polymeric nanoparticles with an

aim to achieve targeted delivery of paclitaxel. Nanoparticles were developed by using

biodegradable methoxy poly(ethylene glycol)-poly-(ε-caprolactone) (MPEG-PCL)

diblock copolymer. Paclitaxel loaded nanoparticles had shown very high entrapment

efficiency above (95%) and sustained release during in vitro experiments. The maximum

tolerated dose (MTD) of paclitaxel loaded nanoparticles after single dose in Balb/c mice

was above 80 mg PTX/kg body weight (b.w), which was 2.6-fold higher than that of

Taxol(R)

(30 mg paclitaxel/kg b.w). The higher concentration of paclitaxel found in tumor

tissue in paclitaxel loaded nanoparticles administered group in comparison to taxol

Chapter 2 - Review of literature

-24-

treated group. It was concluded that nanoparticles based paclitaxel formulation is good

alternative to conventional formulation for controlled delivery of paclitaxel.

Kievit et al. (2011) studied the efficiency of doxorubicin loaded iron oxide

nanoparticles in comparison to doxorubicin drug solution. The results indicated that

doxorubicin nanoparticles were readily taken up by drug resistant cells and greater

reduction in cell viability was found than cell treated with doxorubicin solution. The

results suggest that doxorubicin nanoparticles could improve the efficiency of

chemotherapy.

Kim et al. (2012) developed the nanoparticles that facilitate intracellular delivery

of nanoparticles within the tumor. Hydrophobically modified glycol chitosan

nanoparticles conjugated with interleukin-4-receptor (IL-4R) binding peptides were

developed and were tested in mice bearing positive tumors. The results indicated

enhanced cellular uptake of nanoparticles in tumors in comparison to conventional

approach. Kilicay et al., 2011 developed natural polymer based etoposide loaded

nanoparticles attached with folic acid as ligand. These nano particles found more

effective on HeLa cell line than etoposide loaded plain nanoparticles and shown more

potential as a targeted cancer therapy.

Sheihet et al. (2012) prepared tyrosine derived nanospheres loaded with

paclitaxel and evaluated the toxicity and efficacy of this drug delivery system in

comparison to Cremophor EL based marketed formulation. The results of this study

suggested that nanospheres based formulation significantly increased the maximum

tolerated dose and enhanced anti tumor efficacy in tumor breast cancer cell lines.

The above studies suggested that nanoparticles for drug delivery of anti-cancer

drugs are a useful approach to provide site specific and controlled release of drug at the

target tumor site which ultimately improves the efficiency of chemotherapy at lower

dose.

2.5.2 Emulsion systems

Optimum therapeutic outcomes require not only appropriate drug selection, but

also effective drug delivery to the target site. Emulsions which have oil core, in contrast

Chapter 2 - Review of literature

-25-

to the aqueous core of liposomes, can provide formulations for poorly water-soluble

agents with improved therapeutic efficacy and reduced toxicity (Higashi et al., 1999). For

example, in case of paclitaxel, the drug would be carried in the oil phase since it is poorly

soluble in water and does not possess the amphiphilicity required it to be localized at the

oil-water interface. Emulsions are useful to deliver drug at particular site and helpful to

reduce drug toxicity, provide ease of manufacture and scale-up and low cost as

compared to other colloidal carriers (Floyd,1999). Different types of emulsion systems

are available as drug delivery system like oil-in-water, lipid emulsions, water-in-oil

emulsions, self emulsifying drug delivery systems, lipid nanoemulsions, microemulsions,

solid emulsions, multiple emulsions and modified emulsions. Emulsion systems have

been widely studied for delivery of anti-cancer drugs due to their ability to solubilize

poorly water soluble drugs like paclitaxel. Other advantages associated with emulsion as

carrier is biocompatibility due to use of natural oils, lipids and emulsifiers.

Kan et al. (1999) first time formulated o/w emulsion as the drug carrier which

incorporates paclitaxel in the triacylglycerol stabilized by a mixed-emulsifier system.

Optimized formulation contained 0.75 mg/mL paclitaxel, 10% (w/v) oil blend, 4% (w/v)

egg phosphatidylcholine, 3% (w/v) and Tween 80 in 2.25% (w/v) glycerol solution. The

formulated emulsion exhibited good stability when tested at 4C for three months. In vivo

evaluation of paclitaxel emulsion in ascetic-tumor bearing mice showed the significantly

(p < 0.05) higher anti-cancer activity in comparison to drug solution. Therefore,

formulated emulsion was proposed as promising carrier for sustained delivery of

paclitaxel. In further study, Kang et al., 2004 developed an optimal paclitaxel

microemulsion prepared by self-microemulsifying drug delivery system (SMEDDS)

which is a mixture of paclitaxel, tetraglycol, Cremophor ELP, and Labrafil 1944 and

PLGA. The droplet size for formulated microemulsion was found in nanometers (45-270

nm). The released behavior of paclitaxel from microemulsion containing PLGA having

various molecular weights (8K, 33K, and 90K) exhibited a biphasic release, for first 48 h

initial fast release, followed by a slower and continuous release to 144 h. In contrast,

release of paclitaxel from microemulsion without PLGA was finished during 24 h. The

result was identical with the result of anti-tumor activity in vitro of paclitaxel from micro

Chapter 2 - Review of literature

-26-

emulsion containing PLGA against human breast cancer cell line and this formulation

had shown enhanced anti-tumor activity in vivo compared with micro emulsion without

PLGA.

Yanasarn et al. (2009) developed a lecithin in water emulsion system as an

alternative drug delivery system for docetaxel with aim to improve its efficacy. The

docetaxel encapsulated form was found more effective in killing tumor cells in culture

than free docetaxel. Moreover, the encapsulated nanoparticles were not found to cause

any significant red blood cell lysis or platelet aggregation in vitro, nor did induce

detectable acute liver damage when injected intravenously. Finally, compared to free

docetaxel, the intravenously injected docetaxel nanoparticles increased the accumulation

of the docetaxel in a model tumor in mice by 4.5- fold. Authors hypothesized that these

lecithin-based nanoparticles have potential to be a novel biocompatible and efficacious

delivery system for docetaxel.

Lo et al. (2009) prepared self emulsifying o/w formulations of paclitaxel without

Cremophor EL by using mixed non-ionic surfactants. The surfactants used included

phosphatidylcholine purified from egg yolk (EPC), Tween, and Span. Oils phases were

either pure components or blends from benzyl alcohol, 2-phenylethanol benzyl benzoate,

and tributyrin. Among these surfactants, mixtures of EPC and Tween-80 gave really

stable emulsions in size ranging from 70 to 200 nm. The optimum formulation contains

oils from 1 to 3 wt%, Tween-80 and EPC from 0.4 to 1.2 wt%, respectively.

Consequently, near 500 ppm of paclitaxel can be contained in emulsions. Negligible

cytotoxicity of without drug emulsions assessed with NIH/3T3 cells implied their good

biocompatibility and promising applications as drug delivery carriers.

Wang et al. (2009) prepared a hydroxylcamptothecin (HCPT) anti-cancer drug

emulsion and determined its efficacy in comparison to marketed available injectable

dosage form. Release studies indicated HCPT emulsion exhibited prolonged release

behavior. In vivo study revealed that developed emulsion system has enhanced activity of

drug against cancer.

Chapter 2 - Review of literature

-27-

Wang et al. (2011b) prepared paclitaxel micro emulsion containing reduced

amount of Cremophor EL and evaluated pharmacokinetics, biodistribution and in vivo

anti-tumor efficacy. The antitumor efficacy of the paclitaxel microemulsion in OVCRA-3

and A 549 tumor-bearing animals was similar to that of paclitaxel marketed formulation.

The incidence and degree of allergic reactions exhibited by the paclitaxel microemulsion

group, with or without premedication, were significantly lower than those in the

paclitaxel injection group.

Su et al. (2011) prepared the Vinorelbine-loaded lipid emulsion (VLE) and

compared its toxicity and its anti-tumor efficiency in comparison to conventional

marketed formulation. VLE significantly reduced the toxicity in comparison to marketed

formulation. Comparable anti tumor efficiency was also obtained in comparison to

marketed formulation.

Luo et al. (2012) developed hydroxylcamptothecin (HCPT) loaded emulsion spun

fibers and evaluated anti tumor efficiency by in vitro and in vivo method. In vitro

cytotoxicity tests on HCPT-loaded electrospun fibers indicated over 20 times higher

inhibitory activity against HepG2 cells than free HCPT. Similarly, HCPT-loaded fibers

indicated superior in vivo antitumor activities and fewer side effects than free HCPT. The

above results demonstrate the potential use of emulsion electrospun fibers as drug carriers

for local treatment of solid tumors.

The above studies suggested that emulsion systems are safer to administer and

easier to prepare but some problems such as difficulty in particle size reduction and low

entrapment efficiency limit the application of emulsion systems in drug delivery.

2.5.3 Polymeric micelles

Polymeric micelles are currently recognized as one of the most promising

modalities of drug carriers (Allen et al., 1999; Kataoka et al., 1993; Lavasanifar et al.,

2000). Polymeric micelles have a unique core-shell structure, in which an inner core

serving as a nano container of hydrophobic drugs surrounded by an outer shell of

hydrophilic polymers, such as poly (ethylene blycol) PEG, and have demonstrated

Chapter 2 - Review of literature

-28-

longevity in the bloodstream and effective tumor accumulation after their systemic

administration (Kwon et al., 1994; Nishiyama et al., 2003; Bae et al., 2005). Importantly,

critical features of the polymeric micelles as drug carriers, including particle size,

stability, loading capacity and release kinetics of drugs, can be modulated by the

structures and physicochemical properties of the constituent block copolymers. Also,

polymeric micelles have several advantages, such as a simple preparation method,

efficient drug loading without chemical modification of the parent drug, and controlled

drug release (Kataoka et al., 2001). Polymeric micelles are of particular interest because

of their efficacy in entrapping a satisfactory amount of hydrophobic drugs within the

inner core, their stability in the circulation and their ability to sustain release of drugs. In

addition, the highly hydrated outer shells of the micelles prevent reticuloendothelial

system (RES) uptake and inhibit intermicellar aggregation of their hydrophobic inner

cores. The characteristics of these polymeric micelles could be advantageous for passive

delivery and to extravagate the drug at tumor sites by enhanced permeability and

retention (EPR) effects.

Polymeric micellar formulation of paclitaxel was prepared using AB block of

copolymer of poly (N-(2-hydroxypropyl) methacrylamide lactate-b-polyethylene glycol)

(pHPMAmDL-b-PEG) (Soga et al., 2005). Paclitaxel was found successfully loaded in

the micelles upto 2 mg/mL. Paclitaxel loaded micelles have shown 60 nm mean size with

narrow size distribution and sustained the release of drug to 20 h (70%). Paclitaxel

micellar formulation also showed comparable in vitro cytotoxicity against B16F10 cells

compared to the commercial paclitaxel formulation containing Cremophor EL, while

pHPMAmDL-b-PEG micelles without paclitaxel were far less toxic than the Cremophor

EL vehicle. Above results suggested that pHPMAmDL-b-PEG block copolymer micelles

are a promising delivery system for the parenteral administration of paclitaxel.

Kim et al. (2001) evaluated efficacy, tissue distribution and toxicity of paclitaxel

containing biodegradable polymeric micellar system in comparison to Cremophor EL

based marketed formulation. Polymeric micellar system developed by using a low

molecular weight, nontoxic and biodegradable amphiphilic diblock copolymer,

monomethoxy poly(ethylene glycol)-block-poly(O D,L-lactide) (mPEG-PDLLA) and

Chapter 2 - Review of literature

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paclitaxel. Paclitaxel polymeric miceller system and marketed formulation showed

comparable in vitro cytotoxicity against human ovarian cancer cell line OVCAR-3 and

human breast cancer cell line MCF7. The Maximum tolerated dose (MTD) for polymeric

miceller system and marketed formulation in nude mice was determined and found to be

60 and 20 mg/kg, respectively. The median lethal dose (LD) in Sprague-Dawley rats was

205.4 mg/kg for polymeric miceller system, while 8.3 mg/kg for marketed formulation.

The biodistribution of paclitaxel after administration of polymeric miceller system

showed 2 to 3-fold higher concentration of paclitaxel in tumor tissue as compare to

marketed formulation. The in vivo antitumor efficacy of polymeric miceller system as

measured by reduction in tumor volume of SKOV-3 human ovarian and MX-1 human

breast cancer implanted in nude athymic mice was significantly greater than that of

marketed formulation.

Hana et al. (2009) reported folate-conjugated polymer micelles synthesized by

mixing folate-poly(ethylene glycol)-distearoylphosphatidylethanolamine (FA-PEG-

DSPE) and methoxy-poly(ethyleneglycol)-distearoylphosphatidylethanolamine (MPEG-

DSPE) to encapsulate anti-cancer agent 9-nitro-camptothecin (9-NC). Authors

investigated the targeting ability of folate-conjugated polymeric micelles against three

kinds of tumor cell lines (HeLa, SGC7901 and BXPC3) and found better uptake of drug

due to folate receptor mediated endocytosis.

Gill et al. (2011) developed paclitaxel loaded lipid based PEG 5000-DSPE

micelles for sustained delivery of paclitaxel and studied tissue distribution, plasma

pharmacokinetics and toxicological evaluation. Paclitaxel was successfully formulated in

PEG-lipid micelles with encapsulation efficiency of 95%. This formulation exhibited a

sustained release of drug in simulated lung fluid. This formulation also exhibited 3-fold

higher accumulation of paclitaxel in lungs in comparison to marketed Cremophor EL

based formulation Taxol. A very low concentration of paclitaxel found in non target

organs with micelles. Finally toxicity results showed that no significant increase in levels

of lung injury found in comparison to normal saline treated group.

Chapter 2 - Review of literature

-30-

Lee et al. (2011) prepared Docetaxel-loaded methoxy-poly(ethylene glycol)-

block-poly(d, l-lactide) (m PEG-PDLLA) micellar formulation and its pharmacokinetics,

efficacy, and toxicity were evaluated in comparison with marketed paclitaxel

formulation Taxotere® in preclinical studies. Results of study indicated that prepared

micellar formulation reduces side effects while retaining anti-tumor efficiency in cancer

patients in comparison to Taxotere®.

The above studies indicated that polymeric micelles are good colloidal

nanocarriers for the targeting of poorly water soluble drugs. Due to their hydrophilic shell

and small size they can accumulate in tumoral tissues.

2.5.4 Solid lipid nanoparticles (SLN)

SLN have been introduced as a NDDS for delivery of drugs in various application

routes (Müller et al., 2000). Since, the beginning of the nineties attention from various

research groups has focused on an alternative to polymeric nanoparticles, the solid lipid

nanoparticles. SLN consist of drug trapped in biocompatible lipid core and surfactant at

the outer shell, offering a good alternative to polymeric systems in terms of lower toxicity

(Khurana et al., 2009). Moreover, the production process can be modulated for desired

drug release, protection of drug degradation and avoidance of organic solvents. This

flexibility in large scale may have a paramount importance in commercialization of new

products (Wissing et al., 2004).

First report for the use of solid lipid nanospheres (SLNs) as carrier for delivery of

paclitaxel was came before a decade when Cavalli et al. (2000) developed stealth and

non-stealth SLNs as colloidal carriers for paclitaxel delivery. Formulation contained

bioacceptable and biodegradable lipids, tripalmitin and phosphatidylcholine, and

incorporate amounts of paclitaxel upto 2.8%. Stealth and non-stealth loaded SLNs were

in the nanometer size range and can be sterilized and freeze dried. Thermal analysis

showed that drug was not crystallize in the SLNs. Release kinetics of paclitaxel from

SLNs showed first pseudo zero order and the amount of paclitaxel released over time was

very low when administered intravenously. Authors concluded that SLNs could therefore

be considered as a slow releasing carrier for delivery of paclitaxel.

Chapter 2 - Review of literature

-31-

Serpe et al. (2004) investigated SLN carrying doxorubicin and paclitaxel for anti

tumor activity of SLN formulations in comparison to conventional marketed formulations

on HT-29 cells. The 50% inhibitory concentration IC50 values were interpolated from

growth curves obtained by trypan blue exclusion assay. In vitro cytotoxicity of SLN

carrying doxorubicin was higher than that of conventional drug formulations.

Intracellular doxorubicin was double after 24 h exposure to loaded SLN versus the

conventional drug formulation, at the highest concentration evaluated by flow cytometry.

In vitro cytotoxicity of paclitaxel-loaded SLN and conventional drug formulation were

similar. It was suggested that SLN could be proposed as alternative drug delivery system

for the delivery of anti-cancer agents.

Zhenghong et al. (2009) studied docetaxel-loaded hepatoma-targeted solid lipid

nanoparticle (tSLN). The cellular cytotoxicity, cellular uptake, subcellular localization, in

vivo toxicity, therapeutic effect, biodistribution and histology of tSLNs were investigated.

The tSLNs was found to have the particle size about 120 nm with higher encapsulation

efficiency > 90%, a low burst effect within the first day and a sustained release for the

next 29 days in vitro. The tSLNs also showed better tolerant and antitumor efficacy in

murine model bearing hepatoma. The histology demonstrated that tSLNs had no

detrimental effect on both healthy liver and liver with fibrosis. These results implied that

this targeted nanocarrier of docetaxel could enhance its antitumor effect in vivo with low

systemic toxicity for the treatment of locally advanced and metastatic hepatocellular

carcinoma (HCC). Not much work has been done using SLN as carrier for anti-cancer

delivery but it is expected that drug delivery scientists will explore this as potential

carrier in future due to lesser toxicity, easy preparation method and highly lipophilic

nature of commonly used anti-cancer drugs.

Jain et al. (2010) prepared and investigated tumor targeting potential of surface

tailored solid lipid nanoparticles (SLNs) loaded with anti-cancer drug doxorubicin.

Results revealed that formulation exhibited a biphasic pattern characterized by initial

rapid release of the drug followed by slow and prolonged release. Significantly higher

cytotoxicity of doxorubicin loaded SLNs found in comparison to doxorubicin drug

Chapter 2 - Review of literature

-32-

solution in A549 cell line. The biodistribution profile exhibited that SLNs were able to

deliver a higher concentration of doxorubicin in tumor mass.

Teskac and Kristi (2010) prepared solid lipid nanoparticles loaded with a

promising chemo preventive drug Resveratrol. The results indicated that intracellular

delivery of drug significantly increased with this carrier system in comparison to drug

solution and finally enhanced cytostatic effect was obtained.

Administration of anti cancer drugs by using solid lipid nanoparticles is a

promising approach. Many problems in the administration of anti cancer drugs like non

target organ toxicity, pitiable specificity and high incidence of drug resistant tumor cells

are at least partially overcome by delivering anti-cancer drugs by using solid lipid

nanoparticles.

2.5.5 Liposomes

Liposomes have been recognized as an effective drug delivery system since its

invention (Bangham and Horne, 1964; Gregoriadis et al., 1976a). Liposomes are micro-

particulate or colloidal carriers, usually 0.05-5.0 μm in diameter which form

spontaneously when certain lipids are hydrated in aqueous media (Gregoriadis et

al.,1976b). Liposomes were considered a drug delivery system of choice for

systemic applications of anti-cancer agents due to colloidal size, easily controllable

surface and membrane properties, large carrying capacity and biocompatibility.

Liposomes are composed of relatively bio- compatible and biodegradable material,

and consist of an aqueous volume entrapped by one or more bilayers of natural

and/or synthetic lipids. Drugs with widely varying lipophilicities can be

encapsulated in liposomes, either in the phospholipid bilayer, in the entrapped

aqueous volume or at the bilayer interface. Currently doxorubicin is commercially

available as liposomal formulations (Table 2.1). This formulation have advantages of

reduced cardiotoxcity, increased blood levels and enhanced circulation time and

maximizing drug accumulation at tumor sites. Lot of research is going on use of

liposomes for systemic delivery of anti-cancer drugs. Liposomes has been widely studied

for delivery of anti-cancer drugs with objective of increasing solubility, sustained and

Chapter 2 - Review of literature

-33-

targeted release and enhanced tumor accumulation (Tables 2.4-2.6). The current section

summarized some important findings of use of liposomes as carrier for anti-cancer drug

delivery.

2.5.5.1 Liposomes for sustained and targeted delivery of anti-cancer drugs

5-Fluorouracil (5-FU) is highly hydrophilic anti-cancer drug and its liposomal

formulation has problem of poor encapsulation efficiency and drug leaking during

storage. Vesicular phospholipid gels (VPG) which is highly concentrated liposomal

dispersions contain high amount of phospholipids (30% w/w) for 5-FU delivery was

prepared and investigated (Kaisera et al., 2003). This formulation was found to solve the

problem of poor entrapment efficiency of 5-FU. This formulation was prepared by high

pressure homogenization techniques. The entrapment efficiency of formulation was

found approximately 40% after redispersion of the gel to a liposomal dispersion. The

results of in vitro drug release study at pH 8.0 showed the initial higher release for first

20 min followed by sustained release to 6 h period of time. Author suggested that 5-FU

loaded VPG could be used as implants for sustained release of 5-FU.

Potential lipophilic prodrug of 5-fluorouracil-N3-O-toluyl-fluorouracil (TFu) was

synthesized and liposomal formulation was prepared with objective to improve the

bioavailability and therapeutic efficacy of 5-FU by oral and intravenous administration

(Weitong et al., 2008). Dramatically increased in entrapment efficiency of TFu in

comparison to hydrophilic 5-Fu in liposomal formulation was found. In vitro drug release

profile of TFu-loaded liposomes demonstrated that liposomal formulation followed bi-

exponential equation initial higher release followed by slow release. Pharmacokinetic

studies showed bioavailability of TFu-loaded liposomes was 2.0 fold higher than the

suspension after oral administration, and was bioequivalent comparing with TFu 50%

alcohol solution after intravenous (i.v.) administration. Authors hypothesized that TFu-

loaded liposomes can develop as alternative for oral and i.v. administration

Hao et al. (2005) studied fluid and solid liposomal formulations of topotecan

(TPT) with different composition for in vitro stability and biodistribution behavior.

Authors found that compared with the 'fluid' liposome (S-Lip) composed of soybean

Chapter 2 - Review of literature

-34-

phosphatidylcholine (SPC), the 'solid' liposome (H-Lip) composed of hydrogenated

soybean Phosphatidylcholine decreased the leaking efficiency of TPT and enhanced the

in vivo stability of liposome. The results of biodistribution studies in S180 tumor-bearing

mice showed 5 and 19 fold increase in the TPT area under curve AUC for S-Lip and H-

Lip formulation, respectively. PEG-modified H-Lip (H-PEG) showed 3.7-fold increase in

AUC compared with H-Lip, but there was no significant increase in tl/2 and AUC for

PEG-modified S-Lip (S-PEG) compared with S-Lip. Authors hypothesized that

membrane fluidity of liposome has an important effect on in vitro stability and in vivo

biodistribution pattern of liposomes containing TPT, and PEG-modified 'solid' liposome

may be an efficient carrier of TPT.

Lyophilized negatively charged paclitaxel magnetic liposome was studied as a

potential carrier for sustained and targeted delivery to breast carcinoma via parenteral

administration (Zhang et al., 2005). Encapsulation of paclitaxel in magnetoliposomes

produced significant difference in pharmacokinetic over the drug in Cremophor

EL/ethanol with an increased t1/2 to 19.4 h against 4.1 h. The biodistribution pattern was

also found to significantly higher in tumor tissue with magnetoliposomes than lyophilized

conventional liposomes or Cremophor EL/ethanol. This study demonstrated that

paclitaxel magnetoliposomes can effectively delivered to tumor and exerted a significant

anti-cancer activity with fewer side effects in the xenograft model.

Vodovozova et al. (2007) synthesized a lipid conjugate of the anti-cancer agent

methotrexate (MTXDG) and found that the conjugate successfully encapsulated in the

lipid bilayer of liposomes. The liposomal formulation of MTXDG was found to

overcome the resistance of tumor cells in vitro to methotrexate. Authors have performed

the cytotoxic activities (IC50) of MTX in cultures of the human T-lymphoblastic leukemia

cell line CEM-CCRF and the MTX-resistant subline CEM/MTX and found better anti-

cancer activity of developed formulation.

Urbinati et al. (2010) prepared liposomes containing histone deacetylase

inhibitors (HDACi) and optimized the formulation. They have evaluated the cell viability

of developed formulation in breast cancer cell lines SKBR3 and MCF-7 by administering

Chapter 2 - Review of literature

-35-

without drug loaded liposomes and drug loaded formulation. The observed results

indicate that no cytotoxicity of unloaded liposomes and altered breast cancer cell viability

found with drug loaded liposomes in comparison to drug solution.

Chang et al. (2010) optimized the liposomes for the administration of

mitoxantrone (MTO) with the aim to improve the therapeutic effect of drug. The anti-

cancer activity was evaluated in peritoneal carcinomatosis model. This system exhibited

the strongest binding affinity for MTO, the highest anti-cancer activity and the lowest

toxicity. This cardiotoxcity of MTO was significantly reduced in comparison to drug

solution.

Naik et al (2012) prepared the RGD grafted docetaxel liposomes and evaluated in

vitro cytotoxicity, mechanism of cell death, in vivo pharmacokinetic and biodistribution

behavior of formulation. The results indicated sustained intracellular release of drug from

liposomal system with site specific distribution of drug to tumor and enhanced anti tumor

activity.

Biswas et al. (2012) synthesized a novel polyethylene glycol-

phosphatidylethanolamine (PEG-PE) conjugate with the Triphenylphosphonium (TPP)

group attached to the distal end of the PEG block (TPP-PEG-PE). This conjugate was

incorporated into the liposomal lipid bilayer, and the modified liposomes were studied for

their toxicity, mitochondrial targeting, and efficacy in delivering paclitaxel (PTX) to

cancer cells. PTX-loaded TPP-PEG-L demonstrated enhanced PTX-induced cytotoxicity

and anti-tumor efficacy in cell culture and mouse experiments compared to PTX-loaded

conventional liposomes.

2.5.5.2 Liposomes for increasing the solubility of anti-cancer drugs

First time, Jubo et al (2006) prepared the cholesterol-free liposome formulation

from the mixtures of egg phosphatidylcholine (EPC) and poly (ethylene glycol)

conjugated distearoyl phosphatidylethanolamine (DSPE-PEG 2000) for delivery of a

novel anti-cancer agent ML220 (2-(5-bromo-1H-indol-3-yl)-1H-phenanthro[9,10-d]

imidazole). ML220 is a highly lipophilic drug with a water solubility of 0.14 g/mL and

Chapter 2 - Review of literature

-36-

calculated log P of 5.69. The liposomal formulation found to 50,000 fold increased in

water solubility of drug with loading efficiency of 83%. Evaluation of the subacute

toxicity of the liposome formulated drug in C3H mice revealed no overt signs of toxicity.

Also, a biexponential drug plasma concentration pattern was found upon evaluation of

the pharmacokinetics in Balb/C mice. The in vivo evaluation of the anti-cancer activity in

a human colon HT29 carcinoma model revealed a significant delay in tumor growth. This

study highlighted the potential of cholesterol-free liposomes as a formulation strategy for

highly lipophilic drugs like ML220 and paclitaxel. In similar studies, Zhang et al., 2004

reported the significant increased in solubility and encapsulation efficiency of new anti-

cancer agent Camptosar(R)

(SN-38) in liposomal formulation. SN-38, 7-ethyl-10-

hydroxycamptothecin, is the active metabolite of Irinotecan (CPT-11) and is 200-2000

fold more cytotoxic than irinotecan. Despite its promising anti-cancer potential, SN-38

thus far has not been used as an anti-cancer drug due to its poor solubility in any

pharmaceutically acceptable solvents.

Yang et al (2007a) prepared and evaluated liposomal formulation of paclitaxel.

The results showed that 5% (v/v) of polyethylene glycol 400 in the hydration medium of

liposome significantly increased the solubility (up to 3.39 mg/mL) as well as the EE and

the paclitaxel content in the liposome formulation composed of 10% (w/v) of S100PC

with cholesterol (cholesterol-to-lipid molar ratio = 10:90). When sucrose (sugar-to-lipid

molar ratio = 2.3) was added as a lyoprotectant during the freeze-drying of the liposome,

physicochemical stability of liposome was significantly improved. The cytotoxicity of

liposomal formulation against MDA-MB-231 human breast cancer cell line was not

significantly different in comparison to marketed formulation.

2.5.5.3 PEGylated liposomal formulation

PEGylation of liposomal formulation was found to avoid rapid clearance by

reticuloendothelial system (RES), thus allowing them to remain in the circulation for

prolonged periods after administration. The use of PEGylated liposomes also resulted in

favorable pharmacokinetics of the potential therapeutic agent. These properties of

PEGylated liposomes results in effective tumor targeting and therapeutic efficacy in

number of studies.

Chapter 2 - Review of literature

-37-

Table 2.5: NDDSs reported for increasing the solubility of anti-cancer drugs.

Drug Name System Findings Reference

Paclitaxel Nano particles High encapsulation

efficiency

Enhanced solubility

Mu and

Feng, (2003)

Paclitaxel Nanospheres Prolonged release of

paclitaxel upto 3 months

Enhanced solubility

Feng et

al.,(2002)

ML 220 Liposomes 50,000 fold increase in

the water solubility.

Jubo et

al.,(2006)

SN-38 Liposomes Enhanced entrapment

efficiency upto 95%.

Zhang et al

(2004)

Paclitaxel Liposomes Enhanced solubility Yang et al.

(2007a)

Paclitaxel Polymeric Nanoparticles Enhanced entrapment

efficiency

Wang et

al.,(2011a)

Pakunlu et al. (2006) studied cellular uptake of conventional and PEGylated

liposomes and found that liposomes can be successfully used both for cytoplasmic and

nuclear delivery of anti-cancer drugs. Authors tested PEGylated liposomes of

doxorubicin (DOX) and found that encapsulation of DOX into liposomes substantially

increased the in vitro cytotoxicity and in vivo anti-tumor activity. In subsequent, study Li

et al., 2008 encapsulated Mitoxantrone (MIT) into 60, 80 and 100 nm PEGgylated

hydrogenated soya phosphatidylcholine/cholesterol (HSPC/chol) vesicles using a

transmembrane (NH4)2SO4 gradient method. In vitro release studies revealed that small-

sized formulation had fast drug-release rate. Acute toxicity studies performed in C57

mice proved that PEGgylated liposomal MIT formulations were well-tolerated at a dose

of 9 mg/kg in comparison, severe toxicity induced by free MTO in comparable

concentration.

Yang et al. (2007b) compared the PEGylated immunoliposomes and PEGylated

liposomes for targeted delivery to human breast cancer cells using receptor-mediated

endocytosis. The PEGylated immunoliposome showed substantially higher cellular

Chapter 2 - Review of literature

-38-

uptake than the PEGylated liposome in cancer cells (BT-474 and SK-BR-3) over-

expressing human epidermal growth factor receptor 2 (HER2) at 37 °C, while no

difference was found in low HER2 expressing cells (MDA-MB-231). Pharmacokinetics

of paclitaxel in the PEGylated immunoliposome was compared with that in Taxol(R)

and

in the PEGylated liposome in rats. The circulating time of paclitaxel in the PEGylated

immunoliposome was prolonged compared to Taxol(R)

while slightly shortened than that

in the PEGylated liposome. It was hypothesized that paclitaxel-loaded PEGylated

immunoliposome using Herceptin could serve as a promising model for future tumor

specific cancer therapy of HER2 over-expressing breast cancers.

Yoshizawa et al. (2011) formulated PEG liposomes as drug carrier for the

delivery of paclitaxel and determined the in vitro release and in vivo efficacy of

formulated formulation in comparison to paclitaxel loaded conventional liposomes. The

results of the study confirmed that paclitaxel -PEG liposomes delivered significantly

larger amount of paclitaxel to tumor tissue and provide more excellent anti-tumor effect

than paclitaxel conventional liposomes.

The above studies in which liposomal drug delivery systems were studied

indicated that this approach has provided an opportunity to enhance the therapeutic

efficiency of drugs by altering their solubility and biodistribution. Some studies

suggested that liposomal systems significantly increased the cytotoxicity of the anti

cancer drugs when administered by using liposomes.

2.5.6 Miscellaneous approaches

Hureaux et al. (2010) studied toxicological behavior of blank and paclitaxel-

loaded LNCs after i.v administration in mice. Paclitaxel-loaded LNC formulation was

given i.v. at the dose of 12 mg/kg per day for 5 consecutive days in comparison with

blank LNCs and saline. No mortality was observed in repeated injections studies.

Histological studies revealed no lesions and no accumulation of lipids and blood

parameters of treated animals were found to be normal. The tumoral growth was

significantly reduced in the group treated by paclitaxel-loaded LNCs. The MTDs/LD50s

of marketed Cremophor EL based formulation, paclitaxel-loaded LNCs and blank LNCs

Chapter 2 - Review of literature

-39-

were 12/19.5, 96/216 and above 288/288 mg/kg, respectively. This study demonstrated

that a five-day i.v. injection schedule of paclitaxel-loaded LNC dispersions induced no

histological or biochemical abnormalities in mice and improves paclitaxel efficacy and

therapeutic index in comparison to marketed formulation.

Lacoeuille et al. (2007) investigated the therapeutic efficiency of paclitaxel

loaded LNCs in chemically induced HCC model in wistar rats in comparison to

conventional marketed paclitaxel formulation and controls. Survival curves of paclitaxel

treated groups showed a statistical significant difference compared to the control survival

curve. Animals treated with 4×70 mg/m2 of paclitaxel-LNCs showed the most significant

increase in mean survival times and cases of long-term survivors were preferentially

observed in the paclitaxel-LNCs treated group compared to the controls. These results

demonstrated the great interest to use LNCs as drug delivery system for paclitaxel,

permitting with an equivalent therapeutic efficiency to avoid the use of toxic excipients

such as polyoxyethylated castor oil for its formulation.

Burger et al. (2002) developed cisplatin lipid nanocapsules which overcomes the

problem of solubility of cisplatin. The results of cytotoxicity study in comparison to free

drug solution revealed 100folds higher cytotoxicity.

Garcion et al. (2006) studied the efficacy of paclitaxel lipid nanocapsules in

comparison to commercial Cremophor EL based formulation. The results indicated that

paclitaxel loaded lipid nanocapsules were more efficient than commercially available

formulation for clinical use, thus reducing tumor expansion in vitro and in vivo.

Park et al. (2009) investigated toxicity of Cremophor EL free formulated

paclitaxel solid dispersion. The results revealed that there were no remarkable clinical

signs or deaths related to paclitaxel solid dispersion even at doses upto 160 mg/kg of

paclitaxel. But Taxol(R)

resulted in clinical signs when it contained more than 30 mg/mL

paclitaxel. The LD50 for paclitaxel solid dispersion was above 160 mg/kg and the LD50

for Taxol(R)

was 31.3 mg/kg, more than 5 times lower than that of paclitaxel solid

dispersion. Nephrotoxicity potential of paclitaxel soild dispersion in comparison to

marketed formulation was significantly low. Paclitaxel solid dispersion showed about

Chapter 2 - Review of literature

-40-

10% hemolytic activity, whereas marketed formulation showed about 40% hemolytic

activity when it contained 2 mg of paclitaxel. It was revealed that Cremophor-free

paclitaxel solid dispersion as an injectable formulation is a promising approach to

increase the safety and clinical efficacy of paclitaxel for treatment of cancer.

Liu et al. (2012) prepared solid dispersion of paclitaxel without using Cremophor

EL and evaluated pharmacokinetics, tissue distribution and anti-tumor efficacy in

comparison to marketed formulation. Paclitaxel solid dispersion shows enhanced

efficacy, LD50 and better pharmacokinetic behavior in comparison to commercial

formulation.

As discussed in above section different drug delivery systems have their own

advantages and limitations for developing the commercial viable formulation for anti-

cancer drug delivery. Table 2.7 summarizes the advantages and limitations of different

novel drug delivery systems for anti-cancer drug delivery.

2.5.7 Localized drug delivery of anti-cancer agents

The new innovations in cancer chemotherapy are the development of drug

delivery systems which targets the cancer cells and leave normal cells undamaged.

Localized drug delivery is a way to deliver drug from a dosage form to a particular tumor

site where its entire pharmacological effect is desired. The localized or targeted delivery

of chemotherapeutics has been exploited to limit the indiscriminate toxicities to normal

tissues. Local administration of chemotherapeutics at the tumor site is also thought to

enhance the chemo responsiveness by exposing tumors and adjacent metastases to high

local concentration of anti-cancer drugs. The life time probability of developing an

invasive cancer is 44% for men and 38% for women (Jemal et al., 2010). Drugs

administration by conventional dosage forms such as oral or i.v administration creates a

burden on the whole body system, while the requirement is only at a particular cancer

site. Moreover, conventional routes are not effective over a length of time due to facile

metabolism and repeated administration with high doses is required, which is not

convenient for patients. In last decade, constant efforts have been made to reduce the

Chapter 2 - Review of literature

-41-

Table 2.6: NDDSs for sustained and controlled delivery of anti-cancer drugs.

Drug Name System Finding Reference

Paclitaxel Nano particles 13.11 times higher

therapeutic effects than

Commercial taxol

formulation

Sun et al.,(2008)

Paclitaxel Nanospheres Sustained release of

paclitaxel upto 20 h.

Feng et al.,(2002)

Paclitaxel Polymeric Micelles Sustained release of

paclitaxel upto 20 h.

Soga et al.,(2005)

Paclitaxel Solid Lipid Nanospheres Sustained delivery of

paclitaxel.

Cavalli et

al.,(2000)

5- FU Vesicular phospholipid

gels (VPG) 96% drug released in 100

h and showed 4.5 fold

increase in half life

Kaisera et

al.,(2003)

Paclitaxel Nanoparticles 81.6% cumulative

paclitaxel was released in

30 days and showed

sustained release

properties.

Xua et al.,(2005)

Paclitaxel PEGylated Immuno

Liposomes Biological half life

increased from 5.05 h to

17.8 h

Yang et

al.,(2007b)

Paclitaxel Emulsion system Slow and sustained release

of drug in comparison to

commercial Taxol

formulation.

Panayiotis et

al.,(2002)

Paclitaxel PEG Liposomes Enhanced AUC of

paclitaxel

Yoshizawa et

al.,(2011)

Paclitaxel PEG 5000-DSPE micelles Sustained release of

paclitaxel

Enhanced efficacy

Gill et al.,(2011)

Paclitaxel Polymeric micellar system Higher MTD

Enhaced anti-tumor

activity

Kim et al.,(2001)

Paclitaxel Microparticles Sustained release of drug Chakravarthi et

al.,(2010)

Paclitaxel Microemulsion Low hemolytic toxicity

Enhanced cytotoxicity

Wang et al.,2011b

Doxorubicin Solid lipid nanoparticles Enhanced anti-tumor

activity

Serpe et al.,(2004)

Chapter 2 - Review of literature

-42-

Table 2.7: Advantages and limitations of different novel drug delivery systems for anti-cancer drug delivery.

Carrier Description Advantages Limitations Reference

Liposomes Consisting of lipid

bilayer with aqueous

interior. Generally

phospholipid

Biocompatible and altered the biodistribution of

encapsulated anti-cancer drug and reduces its

toxicity.

Accumulate both hydrophilic and lipophilic drugs.

High drug loading capacity.

Poor storage stability.

Batch to batch reproducibility.

Low drug entrapment.

Particle size control.

Gregoriadis,

1976a and

1976b,

Jubo et al.,

2006

Nanoparticles Particulate carrier in

size range of 10 to

1000 nm. Made from

wide variety of

polymers

Pass through the smallest capillary.

Easily penetrate cells and tissue gap to arrive at

target organs.

Controlled release properties of the drug.

Reduce toxic side effects.

Difficult handling in liquid and dry

forms.

Limited drug loading and burst

release.

Rapid clearance from systemic

circulation by mononuclear

phagocytes system (MPS).

Hamidi et

al., 2008,

Vasir et al.,

2005

Solid Lipid

Nanoparticles

Crystalline or semi-

crystalline stabilized

by surface coating.

Suitable for highly lipophilic anti-cancer drugs like

paclitaxel.

Enhancement of bioavalibility of entrapped drug.

Improvement of tissue distribution and targeting of

drugs.

SLN shown wide application spectrum.

Particle growth.

Particle aggregation.

Unpredictable gelation tendency.

Limited drug loading capacity.

Polymorphic transformation of the

lipid crystal.

Muller et

al., 2000,

Khurana et

al., 2009

Emulsion

systems

Made from a variety

of lipids or other

polymers, droplet size

an order of 100 nm in

case of nanoemulsion.

Solubilize considerable amount of lipophilic drugs.

Ease of manufacturing and scale up.

Low cost as compare to other colloidal systems.

Spontaneity of formation.

Suitable for oral, dermal, ocular and parenteral

delivery.

Poor physical stability.

Risk of emboli formation.

Need strict aseptic handling.

Rapid growth of microorganisms.

Floyd, 1999,

Kang et al.,

2004

Polymeric

micelles

Droplets of surfactants

(lipid or biopolymers)

in a liquid

Simple preparation method.

Efficient drug loading without chemical

modification of parent drug.

Efficient controlled release of drugs.

Precipitation of solubilized drug. Allen et al.,

1999, Kwon

et al., 1994,

Kataoka et

al.,2001

Chapter 2 - Review of literature

-43-

adverse effects of drugs and to increase their therapeutic efficacy by improving drug

delivery systems which localize the anti cancer drugs to the tumor site. Chemotherapy is

most commonly used treatment option for intermediate and late stage cancers. many anti-

cancer drugs have limitation of low aqueous solubility, short biological half life and

narrow therapeutic index due to this reason intravenous administration of drug is not

possible unless formulated in a surfactant containing solution or chemically modified as a

soluble pro-drug. The best example is the use of Cremophor EL in preparation of

paclitaxel formulation. However these strategies can lead to sensitization reactions and

other toxic effects. Furthermore, this intravenous systemic chemotherapy is not

specifically reached to the cancer site, it is very difficult to achieve therapeutic levels of

drug within or adjacent to the tumor and ultimately these limitations reduces the overall

efficiency and safety of chemotherapy. Mielke et al. (1996) reported that with paclitaxel

intravenous therapy almost 50% administered dose eliminated during first 24h, with less

than 0.5% of the total dose locally available to treat tumor. In cancer treatment, failures

due to conventional formulations and high loco regional recurrence rates are indicative of

the shortcoming with current available treatment therapy. The potential benefits of

localized chemotherapy suggested that this type of chemotherapy will improve the

efficacy of treatment and reduce the patient morbidity. The principle of localized drug

delivery by taking example of in situ thermosensitive hydrogel system of anti-cancer drug

is explained in Figure 2.4.

Chapter 2 - Review of literature

-44-

Figure 2.4: Localized drug delivery for anti cancer drugs (A) Loading of drug or carrier system in localized drug delivery system (B) Administration of hydrogel into tumor tissue

2.5.7.1 Advantages of localized and targeted drug delivery

Localized chemotherapy offer following advantages over conventional systemic delivery

(Shackney et al., 1978, Meyer, 1981).

1. Local drug delivery is advantageous over systemic delivery as local drug

concentrations can be maximized in the immediate tumor environment with

prolonged retention time.

2. A lower dose is required in comparison to systemic delivery to fill up the volume of

distribution.

3. This delivery system is beneficial for drugs with dose dependent activity.

4. Some of the therapeutic agents with a relatively short half-life are more effective with

local delivery.

Chapter 2 - Review of literature

-45-

5. After surgery generally local reoccurrence of tumor started at the site of surgical

resection which is one of the major cause of morbidity and mortality clinically.

Delivery of chemotherapeutic drugs after surgery with localized drug delivery

systems will improves the efficacy and reduces the incidence of complications related

to reoccurrence.

6. Direct delivery of drug to the site of cancer, reduces the wastage of drug.

7. Due to sustained and controlled release of drug, frequency of administration can be

minimized.

8. Drugs with low bioavailability can be targeted directly to the required site.

9. Intratumoral delivery is not limited by poor blood supply.

2.5.7.2 Different approaches used for localized drug delivery of anti-cancer drugs

In literature following approaches are reported for localized delivery of anti-cancer drugs

Localized drug delivery of anti-cancer drugs by implantable drug delivery

systems.

Localized drug delivery of anti-cancer drugs by using dermal route.

Localized drug delivery of anti-cancer drugs by using in situ thermosensitive

hydrogel approach.

Localized drug delivery of anti-cancer drugs by using nano carrier approach.

2.5.7.2.1 Localized drug delivery by implantable drug delivery systems

The treatment failure in early and intermediate stage cancers is mainly due to high

reoccurrence rates of cancer cells. To overcome the shortcomings of conventional cancer

chemotherapy, advanced implantable localized delivery systems were developed for the

delivery of anti-cancer drugs (Fung and Saltzman, 1997). Implanting a biodegradable

system loaded with anti-cancer drug near the tumor site provides high concentration of

drug at tumor site which will kill the malignant cells and also prevent the systemic side

effects associated with conventional therapy. Implantable drug delivery systems for

localized delivery of anti-cancer drugs have been embodied in a variety of forms like

drug eluting films, gels wafers, rods and stents. Almost all implantable devices are

Chapter 2 - Review of literature

-46-

biodegradable in nature so there is no need of surgery for device removal. The commonly

used material for the construction of implants is polymers from natural or synthetic

source. Natural polymers those have been investigated for drug delivery application

includes polysaccharides such as alginate, hyaluronic acid, dextran, gelatin and chitosan.

These materials are well tolerated in vivo. Synthetic materials which were investigated

for implants are polycarbonates, polyorthoesters and phosphate based polymers. These

polymers are often hydrophobic in nature and ideally suited for long term delivery. The

major drawbacks to synthetic materials are that many polymers form acidic degradation

products that can accumulate and cause inflammation at the implant site. Implantable

drug delivery systems offer the following advantages over traditional systems.

1. Controlled and prolonged release of drug to ensure adequate diffusion and uptake into

cancer cells over many cycles of tumor cell division.

2. Loading and release of lipophilic anti-cancer drugs are more effective.

3. Stabilization of embedded anti-cancer drug and preservation of anti-cancer activity.

4. Patient compliance more.

5. Less exposure to systemic circulation.

A chemotherapeutic polymer based paste has been reported by Hunter et al., 1997

for application at the time of surgery to reduce local recurrence of disease at tumor

resection sites and a chemotherapeutic polymer coated stent for use in the palliative

management of malignant obstruction to improve the effective lifespan of the device (e.g.

esophageal, biliary, prostate and pulmonary disease).

Shikani and Domb, (2000) developed implantable disks loaded with four anti-

cancer agents (cisplatin, fluorouracil, methotrexate and paclitaxel) for subcutaneous

administration adjacent to tumor, with this system tumor growth was delayed and

reduction in systemic toxicity was found.

Tahar and Ishii, (2001) prepared Apatite cement containing cis-

diaminedichloroplatinum and implanted in rabbit femur and found sustained release of

the anti-cancer drug. Paclitaxel containing chitin and chitin-Pluronic F-108 microparticles

were formulated and characterized as biodegradable systems for localized administration

in solid tumors by Nsereko and Amiji, (2002).

Chapter 2 - Review of literature

-47-

Dhanikula and Panchagnula, (2004 and Ho et al., 2005 have studied the chitosan

based implantable delivery system for local delivery of paclitaxel. Results revealed that

implantable drug delivery system sustained the drug release and paclitaxel exhibits dose

dependent antiangiogenic, antimetastatic and apoptotic properties to block multiple

pathways involved in the growth and spread of cancer.

Ortiz et al. (2007) developed biodegradable Doxorubicin loaded cylinders for

intra-prostatic implantation and evaluated the feasibility of using regional intra prostatic

drug therapy to treat prostate confined cancer.

Emmanuel et al. (2007) developed a novel implantable drug delivery system for

localized delivery of paclitaxel and studied the impact of local sustained delivery on the

development of multi drug resistance. The delivery of paclitaxel with the implant system

did not significantly affect MDR1 expression and finally lower the multi drug resistance.

Soo et al. (2008) studied the localized and sustained delivery potential of a

chitosan-egg phosphatidylcholine (chitosan-e PC) implant system containing PLA-b-

PEG/PLA nanoparticles for the delivery of paclitaxel to treat ovarian cancer. Release of

paclitaxel was found to be sustained over a four-week period following implantation of

the chitosan-e PC system into the peritoneal cavity of healthy Balb /C mice.

Abe et al. (2008) evaluated a hydrohydroxyapatite-alginate composite beads

system for the delivery of paclitaxel in metastatic spine cancer in a rat model. Animals

treated with intra-tumoral injections of the paclitaxel-loaded beads showed a 140-150%

prolongation in disease-free survival compared to intravenous doses of paclitaxel

solution.

Shikanov et al. (2008) had developed paclitaxel loaded biodegradable extended

release implant for localized delivery of drug to solid tumors. Results showed that highest

paclitaxel concentration was 40µg/g of tumor tissue after 1day when administered as

intratumoral injection and decreased gradually after 10 days to 5µg/g, which is still

enough to induce cytotoxic effect and necrotic effect of paclitaxel on the tumors. D--

tocopheryl polyethylene glycol 1000 succinate (TPGS) was used by Dong et al., 2008 as

a novel additive to the poly (L-lactide) (PLLA) films for local drug delivery with

Chapter 2 - Review of literature

-48-

paclitaxel as a prototype therapeutic agent. In vitro results demonstrated sustained release

of paclitaxel from films.

Zhang et al. (2011) formulated modified liposomes with temperature sensitivity

and loaded vinorelbine bitartrate and evaluated the localization potential and anti tumor

effectiveness of formulation. Results of study indicate that significantly higher anti tumor

activity against lung tumor model was obtained in comparison to plain drug solution.

OncoGel, a controlled release depot formulation of paclitaxel also known as

ReGel has been evaluated in vivo to evaluate the efficacy in local tumor management.

The ReGel system water soluble implant comprised of an aqueous solution of

biocompatible polymers. Oncogel showed ability to physically target paclitaxel to the

tumor site with very little reaching in the systemic circulation, resulting in an acceptable

safety profile with dose limiting toxicities being local in nature (Wolinsky et al., 2012).

Table 2.8 summarizes the different drug delivery systems studied for localized delivery

of anti-cancer drugs.

The delivery of anti cancer drugs by using implantable systems suggested that

these systems enhance the concentration of drug at tumor site and improve the quality of

chemotherapy.

2.5.7.2.2 Localized delivery by using dermal route

The drug administration route considerably influences the toxicity and efficacy of

the drug. Generally, anti-cancer drugs are administered by conventional routes.

Conventionally, administered anti-cancer drugs are often extensively distributed to the

normal tissues and only a small fraction of drug reach to the tumor site. This type of

administration reduces the therapeutic efficiency and increase the systemic drug toxicity.

Therefore, poor specificity of anti-cancer drugs poses a significant challenge to cancer

chemotherapy. Moreover, the conventional route (i.v) administration usually requires

hospitalization of patient which can be inconvenient for patient. These shortcomings of

conventional chemotherapy suggests administration of anti cancer drug with new drug

delivery systems, which enhances the localization of drug at tumor site and avoids the

distribution of drug to the normal tissues.

Chapter 2 - Review of literature

-49-

Table 2.8: Different drug delivery systems studied for localized drug delivery of paclitaxel.

Drug Drug delivery system Finding Reference

Paclitaxel Chitosan based

implantable

formulation

Sustained and local

delivery of anti-

neoplastic agent

Ho et al.,(2005)

Paclitaxel Microparticles Localized delivery of

paclitaxel in solid tumors

Nsereko and

Amiji, (2002)

Paclitaxel In situ gel Sustained and targeted

delivery

Jauhari and Dash,

(2006)

Paclitaxel Polymer based paste Reduced re-occurrence of

disease at tumor

resection

Hunter et

al.,(1997)

Paclitaxel Liposomes Increased drug release Rane and

Prabhakar,(2009)

Paclitaxel Controlled release

depot formulation Improved safety profile

of drug

Elstad and

Fowers,(2009)

Paclitaxel Nanoparticles Reduced restenosis Bakowsky and

Kissel,(2007)

Paclitaxel Implant system High concentration in

tumor tissue

Shikanov et

al.,(2008)

Paclitaxel Microbubbles Enhanced anti tumoral

activity

Sustained release

Enhanced entrapment

efficiency

Cochran et

al.,(2011)

Paclitaxel Implant System Localized sustained

delivery of paclitaxel

Emmanuel et

al.,(2007)

Doxorubicin Nanocarrier system Sustained release

Enhanced anti tumor

efficacy

Cai et al.,2010

Paclitaxel Nanoparticles Localized sustained

delivery of paclitaxel

Soo et al.,(2008)

Paclitaxel Composite beads

system Enhanced localized

delivery of paclitaxel

Abe et al.,(2008)

Cisplatin,

methotrexate

fluorouracil

Implantable disks Enhanced anti-tumor

efficacy

Shikani and

Domb,(2000)

Chapter 2 - Review of literature

-50-

As skin is the largest organ of human body and easily accessible, therefore

relatively it is easy to administer anti-cancer drugs through skin. This route provides

several therapeutic advantages such as avoidance of hepatic first pass metabolism, zero

order drug delivery, provide sustained and controlled release of drug, reduced dose

requires for therapeutic effect and localization at the site of action is possible. Anti cancer

drugs are very suitable candidates for delivery through skin because superior and

continuous exposure of drug is required at the site of tumor. Some topical formulations

for localized delivery to skin cancer such as semi-solid formulation of 5-fluorouracil and

photodynamic therapy (PDT) approved by the US Food and Drug Administration

(UDFDA). These therapies are used to treat non melanoma skin cancers but available

conventional approaches have some side effects, such as an intense local inflammatory

reaction, that result in a lack of patient compliance, low penetration of drug through skin.

These drawbacks limit the applicability of this route in the treatment of cancer. Skin is

mainly composed of three primary layers: the epidermis or stratum corneum, dermis and

hypodermis. The stratum corneum plays an important role in the penetration of

substances into the skin and considered as a major barrier for transportation of drugs

through skin. In literature authors reported the different improved strategies for localized

delivery of anti cancer drugs through dermal route. Great efforts have been made to

design topical nanocarriers like liposomes, polymeric micelles and solid lipid

nanoparticles and techniques such as the application of an electric field (e.g.,

iontophoresis and electroporation) that can increase the penetration of anti-cancer drugs

into the deep skin layers (Souza et al., 2011; Araujo et al., 2010; Gelfuso et al., 2008;

Gelfuso et al., 2011) to make topical treatment more effective (Figure 2.5). But all

approaches have some limitations due to which further improvement in these systems

required. These methods have the common goal of overcoming the barrier stratum

corneum and targeting tumor cells efficiently.

Chapter 2 - Review of literature

-51-

Figure 2.5: Methods to improve drug penetration through the skin

In literature many studies have been reported to improve the drug delivery of anti-

cancer drugs through the skin. Glavas-Dodov et al. (2003) has developed liposomal gel

of 5-flurouracil with the objective to deliver drug locally within the skin and found that

liposomal gel system acted as reservoir system for continuous delivery of the

encapsulated drug.

Panchagnula et al. (2004) investigated the applicability of stratum corneum lipid

bilayer alteration by fatty acids and terpenes toward the permeation enhancement of

paclitaxel through rat skin and also studied the feasibility of dermal delivery of paclitaxel

with binary combinations of ethanol and isopropyl myristate. The results indicated that

localization of paclitaxel in the different layers of the skin is possible as per the clinical

need by altering the composition of the solvent system. Similarly, Khandavilli and

Chapter 2 - Review of literature

-52-

Panchagnula 2007 developed paclitaxel loaded nanoemulsion and investigated the

penetration of paclitaxel into deeper layers of skin. The authors found that after dermal

application, the drug was predominantly localized in deeper layers of skin, with minimal

systemic escape.

Curic et al. (2008) prepared topical formulation of invasomes loaded with

tamoxifen mainly used in the treatment skin cancer. The authors investigated the

photodynamic efficacy of prepared topical formulation onto the skin of mice bearing the

subcutaneous implanted human colorectal tumor HT29. The groups of mice treated with

topical formulation showed a significantly reduced tumor as compared to control groups.

Chandrashekar and Shoba Rani, (2009) developed and characterized

microprocessor controlled transdermal delivery of anti-cancer drugs such as 5-

Fluorouracil (5-FU) and 6-Mercaptopurine (6-MP). Results of this study indicated that

flux was significantly increased and it was concluded that transdermal approach for anti-

cancer drugs is effective approach particularly to treat skin cancer.

Suppasansatorn et al. (2007) studied microemulsion as topical delivery vehicle for

the anti-melanoma prodrug, temozolomide hexyl ester. The results revealed that

optimized microemulsion significantly increased the permeation of drug through silicon

membrane and rat skin and it was concluded that this system may be suitable choice for

local delivery of drug in skin cancer.

Shakeel and Ramadan, (2010) developed the nanoemulsion for local delivery of

recently investigated anti-cancer drug caffeine for skin cancer. Result shows that there

was significant increase in permeability parameters in nanoemulsion formulation as

compared to aqueous solution of caffeine and conclusion was made that nanoemulsions

was good carrier for delivery of anti-cancer drug locally.

Zhao et al. (2010) studied transdermal drug delivery system to deliver realgar

nanoparticles and investigated its anti-cancer activity and toxicity potential. Tests on

tumor-bearing C57BL/6 mice displayed that realgar could decrease the tumor volume

markedly via transdermal drug delivery when compared with administration of drug

solution by conventional route.

Chapter 2 - Review of literature

-53-

Ali et al. (2011) developed nanovesicles for topical delivery of 5-flurouracil (5-

FU) and compared its efficiency in comparison to marketed available topical formulation.

The results of study revealed that nanovesicles showed better permeability and retention

than marketed formulation. Improved cytotoxicity was found on HaCaT cell line.

EI Meshad and Tadros, (2011) developed emulsion system for the delivery of

anti- cancer agent 5-FU through dermal route. Author concluded that significant (P<0.05)

increase in 5-FU permeability parameters such as steady-state flux, permeability

coefficient was achieved with optimized formulation in comparison to control solution.

Paolino et al. (2012) developed paclitaxel loaded ethosomes for local treatment of

non melanoma skin cancer. The in vitro data shows that the topical application of

paclitaxel loaded ethosomes improved the permeation of paclitaxel through stratum

corneum and increased its anti-proliferative activity in a squamous cell carcinoma model

as compared to the free drug. Table 2.9 summarized the various approaches reported for

dermal delivery of anti-cancer drugs.

2.5.7.2.3 Localized delivery using thermosensitive hydrogel approach

In the last decade, lot of work has been done on in situ thermosensitive hydrogel

based localized drug delivery systems. These systems are injectable fluids that can be

introduced into the body in minimal invasive manner prior to gelling within the desired

tissue. Gelation can occur generally after a change in pH or temperature. Worldwide,

scientists explore different materials which are used in the preparation of in situ implant

system and thermosensitive nanocarriers. Among those poly (-lactide) and poly (lactide-co-

glycolide) copolymers and complex of poly (methacrylic acid) and poly (ethyleneglycol)

dissolved in a hydro alcoholic solvent were tried by different investigators. By introducing

thermosensitive polymers to prepare in situ thermosensitive drug delivery systems, these

systems possesse the ability of active response to the thermal changes and are biologically

compatible. But all polymers possesses some drawbacks like some polymers require

photopolymerization for which the presence of photoinitiator at the site of gelation required,

some polymers turned into gel at room temperature within 3 h, the organic solvents used to

solubilize some polymers can physically denature the some compounds which are very

crucial for biomedical applications, When precipitation method used for gelation, incomplete

gelation may occurs which may lead to high burst release and can cause local toxicity.

Chapter 2 - Review of literature

-54-

Table 2.9: Approaches reported for dermal delivery of anti-cancer drugs.

To overcome all these drawbacks, some other thermosensitive polymers capable

of forming hydrogel was studied. A lot of work has been done on thermosensitive

polymers like poloxamers and copolymers of N-isopropylacrylamide. But as these

polymers are not biodegradable, weak mechanical strength, and rapid erosion limit their

use (Johnston et al., 1992; Okano et al., 1990). Some authors studied PEG and poly

(lactic acid) block copolymer systems as a thermosensitive combination. These block

copolymers remain in sol state at around 45 oC and gel upon cooling to near about body

temperature (Jeong et al., 1997). But the major drawback of this system is that this

system need to heat to incorporate the drug and to inject the system. As all of these

systems were not fully convincing, other improved systems were investigated that can be

Drug Approach Permeation

barrier Used

Type of

cancer

Reference

5-FU Liposomal gel Rat Skin Skin Cancer Glavas-Dodov et

al. (2003)

Paclitaxel Permeation

enhancers

Rat Skin Breast and

ovarian

Panchagnula et

al. (2004)

Paclitaxel Nanoemulsion Rat Skin Breast and

ovarian

Khandavilli and

Panchagnula

(2007)

Temoporfin Invasomes Rat Skin Skin Cancer Curic et

al.,(2008)

6-Mercaptopurine

and 5-FU

Microprocessor

controlled TDDS

Human Cadaver

skin

Skin Cancer Chandrashekar

and Shoba Rani

(2009)

Temozolomide

hexyl ester

Microemulsion Silicone

membrane and

skin

Skin Cancer Suppasansatorn

et al. (2007)

Caffeine Nanoemulsion Rat Skin Skin Cancer Shakeel and

Ramadan,

(2010)

Realgar Nanoparticles Rat Skin Skin cancer Zhao et al.

(2010)

5-FU Nanovesicles Rat Skin Skin Cancer Ali et al. (2011)

5-FU W/O Emulsion Rat Skin Skin Cancer EI Meshad and

Tadros, (2011)

Paclitaxel Ethosomes Rat Skin Non

melanoma

skin cancer

Paolino et al.

(2012)

5-FU Ethosomes Rat Skin Skin cancer Puri and Jain,

(2012)

Chapter 2 - Review of literature

-55-

used parenterally, capable of forming stable gel within the short period of time after

injection and maintains its physical integrity as long as possible. To achieve these

objectives Ruel-Gariepy et al. (2002) and Chenite et al. (2000) proposed in situ

thermosensitive hydrogel system which contains chitosan and -glycerophosphate that

remains in sol form at 8-15 oC and turns into gel at body temperature upon injecting.

Chitosan is a hydrophilic cationic copolymer produced by the deacetylation of

chitin and composed of glucosamine and N-acetyl glucosamine units. It is a

biodegradable, biocompatible and mucoadhesive biopolymer. This is most commonly

investigated polymer for the preparation of in situ thermosensitive hydrogel system.

Endothermically gelling chitosan solution is prepared by supplementing an aqueous

solution of chitosan with -glycerolphosphate (β-GP) or disodium salt. These solutions

possess a neutral pH; remain liquid at or below room temperature. Upon heating, the

chitosan chains lose their water of hydration, bonding between chains can occur and

gelatin proceeds. Three types of interactions are involved in the gelatin process: (a)

electrostatic attraction between the ammonium groups of chitosan and the phosphate

group of GP, (b) hydrogen bonding between polymer chains as a consequence of reduced

electrostatic repulsion after neutralization of the chitosan solution with GP; and (c)

chitosan-chitosan hydrophobic interactions.

In order to obtain more sustained, controlled and localized effects it is reported by

some groups to load the selected drug in a carrier system like liposomes and then

incorporate chitosan based thermosensitive hydrogel in drug loaded carrier system. As

chitosan is devoid of any surfactant properties the risk of liposomal destabilization is

minimal. Moreover, hydrogel preserve the original structure of liposomes and make the

preparations more user friendly, resulting in better patient acceptability and compliance

(Pavelic et al., 2001).

The main administration routes for the administration of chitosan hydrogel

systems are intratumoral administration and subcutaneous administration next to the

tumor. Obara et al. (2005) administered paclitaxel containing hydrogel subcutaneously

beneath the tumor using 18G needle and disposable syringe. It was reported that hydrogel

Chapter 2 - Review of literature

-56-

formulation strongly inhibited the tumor growth as compared to paclitaxel solution. It is

also reported in literature that 26G needle was suitable for intratumoral administration of

chitosan hydrogel. For administration of hydrogel intratumorally needle is inserted in the

centre of tumor (Ruel-Gariepy et al., 2004; Omelyanenko et al., 1998). After injection,

the needle was held in place for 3-4s before being withdrawn to prevent the hydrogel

from leaking out of the injection site.

Ruel-Gariepy et al. (2002) developed in situ thermosensitive chitosan based

hydrogel containing carboxyfluorescein (CF) loaded liposomes and studied the release

profile and gelation behavior of developed formulation These results indicate that the

liposome-C-GP system rapidly gels at body temperature and can sustain the delivery of

compound over 2 weeks. In another study, Ruel-Gariepy et al. (2004) developed a

thermosensitive chitosan based hydrogel for the local delivery of paclitaxel. The in vitro

release profiles demonstrated sustained release delivery of paclitaxel. Anti tumor activity

experiment showed that one intratumoral injection of the thermosensitive hydrogel

containing paclitaxel was as efficacious as four intravenous injections of marketed

formulation in inhibiting the growth of EMT-6 cancer cells in mice. The developed

formulation was also found less toxic in comparison to marketed formulation.

Berrada et al. (2005) developed thermo responsive non toxic camptothecin

formulation for sustained intratumoral release of anti-cancer agent. This formulation,

containing homogeneously dispersed camptothecin was implanted intratumorally into a

sub-cutaneous mouse tumor model (RIF-l). Animals treated with the polymer implants

containing camptothecin had significantly longer tumor growth delay in comparison to

untreated animals.

Han et al. (2006) developed PNIPAM-AAM based thermosensitive liposomes of

anti-cancer drug doxorubicin with the objective to enhance the release of drug at tumor

site. Thermosensitive liposomes showed much higher levels of tumor growth inhibition in

comparison to doxorubicin free solution.

Jauhari and Dash, (2006) developed in situ gel delivery system for sustained and

targeted delivery of anti-cancer drug. This delivery system consisted of chitosan and

Chapter 2 - Review of literature

-57-

glyceryl monooleate (GMO) in 0.33M citric acid containing paclitaxel. Results of this

study showed sustained and enhanced anti-tumor efficacy of paclitaxel.

Wu et al. (2006) studied the swelling behavior and release profile of doxorubicin

from thermo-and pH sensitive hydrogel composed of quaternized

chitosan/glycerophosphate. . The results of this study suggests that in acidic condition,

the hydrogel dissolves and release drug quickly, while it absorb water and release drug

slowly at neutral or basic conditions.

Shim et al. (2007) developed pH- and temperature-sensitive injectable,

biodegradable block copolymer hydrogel as carriers for paclitaxel. In vitro drug release

and anti-tumor efficiency was evaluated by authors. Release profiles of paclitaxel showed

sustained release of drug. Paclitaxel-loaded block copolymer hydrogel was found to have

good anti-tumor effect for 2 weeks and induced strong apoptosis in tumor tissue.

Zhou et al. (2009) prepared chitosan based in situ hydrogel for the sustained

delivery of Adriamycin. The authors concluded that this system released only 60% of

loaded drug in 24h and sustained the release of drug for longer period of time.

Mulik et al. (2009) developed chitosan based thermosensitive hydrogel containing

liposomes for sustained delivery of anti-cancer agent cytarabine. The physical

characterization of the formulation showed that the system gels at 37 oC within 5 min.

The in vitro and in vivo release study showed that the system can sustain the release of

cytarabine for more than 10 days. In vivo results showed that the AUC and t½ of C-GP

containing cytarabine loaded liposomes is greater in comparison to marketed formulation

and indicated that that drug release rate is slow and drug remains in the body for longer

period of time.

Li et al. (2010) studied gel-sol-gel thermo-gelation behavior of chitosan-inorganic

phosphate solutions. The rheological study results suggest that there are two phase

transition points as in function with temperature. The system is in gel state at 4 ºC. When

the temperature is increased to 30 ºC, the gel-sol transition as well as the decrease in

Chapter 2 - Review of literature

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turbidity was observed. The sol-gel transition as well as the increase in turbidity was

observed again as the temperature was reached at 37 ºC.

The above studies suggested that in situ thermosensitive hydrogel approach has a

potential to deliver chemotherapeutics locally at tumor site. This approach improves the

anti-tumor effect and reduces the systemic toxicity of anti cancer drugs. Table 2.10

summarizes the thermosensitive hydrogel reported for localized delivery of anti-cancer

drugs.

2.5.7.2.4 Localized drug delivery of anti-cancer drugs by nano carrier approach

There are a variety of nanocarrier based drug delivery systems explored by

different scientists for localized delivery of anti-cancer drugs. The main objective of

these systems is to improve overall efficacy of cancer chemotherapy and quality of life by

increasing the bioavailability of drug at tumor site, and low exposure of drug to non

target tissues. The different nanocarrier based localized drug delivery systems were

developed and evaluated. The systemic delivery of nano carriers such as liposomes,

nanoparticles, micelles and dendrimers enhanced the therapeutic efficiency of

chemotherapy by using various approaches such as active or passive targeting to the

tumor, angiogenesis-associated targeting or uncontrolled cell proliferation targeting.

These nanocarriers promise the ability to target tumor tissues with accumulation of

therapeutic concentrations of drug, but sometimes localization is challenging due to

removal of nanomaterials by the reticulo endothelial system.

Bakowsky and Kissel, (2007) revealed catheter-based local delivery of

biodegradable nanoparticles with sustained release characteristics represents a therapeutic

approach to reduce restenosis. To evaluate the anti restenotic effect in vivo, paclitaxel-

loaded nanoparticles were administered locally to the wall of balloon-injured rabbit iliac

arteries using a porous balloon catheter. As a result a 50% reduction in neointimal area in

vessel segments was observed.

In vitro anti-cancer activity of wheat germ agglutinin (WGA)-conjugated PLGA

nanoparticles loaded with paclitaxel and isopropyl myristate was studied by Mo and Lim,

Chapter 2 - Review of literature

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(2005). Intracellular localization, geno- and cytotoxic response of poly N-

isopropylacrylamide (PNIPAM) nanoparticles to human keratinocyte (HaCaT) and colon

cells (SW 480) were investigated by Naha et al. (2010) and suggested that the PNIPAM

nanoparticles showed excellent biocompatibility in vitro.

Ahmed et al. (2006) developed biodegradable polymersomes loaded with

paclitaxel and doxorubicin and evaluated therapeutic efficiency of carriers. A single

systemic injection of the dual drug combination showed a higher maximum tolerated

dose than the free drug cocktail and also shrinks tumors more effectively and more

sustainably than free drug.

Paclitaxel containing liposomes of different phospholipid compositions based

localized drug delivery system was developed by Rane and Prabhakar, (2009). The study

revealed that the formulation composed of Phospholipon 90G/DOPE/CHEMS 8:2:2

containing paclitaxel and lipids in the molar ratio of 1:30 (drug:lipid) was found to have

good incorporation efficiency (94%) and the formulation had shown sustained release of

drug.

Poly(organophosphazene)-paclitaxel conjugate was synthesized by Chun et al.

(2009) by a covalent ester linkage between paclitaxel and carboxylicacid-terminated

poly(organophosphazene). From the in vivo anti tumor activity study with tumor-induced

nude mice, the polymer-paclitaxel conjugate system after local injection at the tumor site

were shown to inhibit tumor growth more effectively and longer than paclitaxel and

saline alone.

Chapter 2 - Review of literature

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Table 2.10: Thermosensitive hydrogels for localized drug delivery.

Drug Drug delivery

system

Hydrogel system used Transition

temp (oC)

Findings Reference

Carboxyfluorescein Liposomes

contained

thermosensitive

hydrogel

Chitosan: βGlycerolphosphate 37 Sustained and controlled

release

Ruel-

Gariepy et

al.,(2002)

Paclitaxel Thermosensitive

hydrogel Chitosan:βGlycerolphosphate 37 4 folds higher inhibition of

tumor in comparison to

conventional injection

Ruel-

Gariepy et

al.,(2004)

Camptothecin Thermo

responsive

hydrogel

Chitosan:βGlycerolphosphate 37 Sustained and localized

intra tumoral delivery of

drug

Berrada et

al.,(2005)

Doxorubicin Doxorubicine

loaded liposomes

in

thermosensitive

hydrogel

Poly(N-isopropylacrylamide-

co-acrylamide) PNIPAM-

AAM

37 Enhanced efficacy in

tumor cells

Han et

al.,(2006)

Doxorubicin Thermosensitve

hydrogel Chitosan:βGlycerolphosphate 37 Sustained release of drug Wu et

al.,(2006)

Paclitaxel Copolymer

hydrogel Biodegradable block copolyner 37 Enhanced anti-tumor

activity

Shim et

al.,(2007)

Cytarabine Cytarabine

loaded liposomes

in

thermosensitive

hydrogel

Chitosan:βGlycerolphosphate 37 Sustained release of drug

Higher t½ and AUC in

comparison to marketed

formulation

Mulik et

al.,(2009)

Adriamycin In situ hydrogel Chitosan:αβGlycerolphosphate 37 Sustained release of

adriamycin

Zhou et al.

(2009)

Chapter 2 - Review of literature

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Cai et al. (2010) prepared biopolymeric carrier system for localized delivery of

doxorubicin for breast cancer. This system exhibits a sustained release characteristic in

vitro and in vivo in the breast tissues of rodents bearing human breast cancer xenografts.

This carrier system e dramatically inhibits breast cancer progression in vivo, leading to an

increased survival rate.

Kaminskas et al. (2011) studied the PEGylated polylsine dendrimers bearing

doxorubicin carrier for tumor targeting. The results indicated that enhanced uptake of

drug into solid tumors at levels 3 to 8 times higher than control but reduced potential for

accumulation in non target organs.

Very recently, Shapira et al. (2011) have exploited nanoparticles for selective

drug delivery to tumor cells. This targeted strategy holds promise in paving the way for

the introduction of highly effective nanoscopic vehicles for cancer therapeutics while

overcoming drug resistance.

2.5.8 Carrier selected for study

Elastic liposomes were developed in order to take the advantage of phospholipid

vesicles for localized drug delivery. These self-optimized aggregates with ultra flexible

membrane are able to deliver the drug reproducibly into or through the skin. These elastic

liposomes are several orders of magnitudes more elastic than the standard liposomes and

thus well suited for the skin penetration. Elastic liposomes overcome the skin penetration

difficulty by squeezing themselves along the intracellular sealing lipids of the stratum

corneum. There is provision for this because of the high vesicle deformability which

permits the entry due to the mechanical stress of surroundings in a self-adapting manner.

Flexibility of elastic liposome membrane is achieved by mixing suitable surface-active

components in the proper ratios (Cevc and Blume, 1992). The resulting flexibility of

elastic liposomes membrane minimizes the risk of complete vesicle rupture in the skin

and allows elastic liposomal formulation to follow the natural water gradient across the

epidermis when applied under non occlusive condition (Cevc et al., 1998).

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The high and self-optimizing deformability of typical composite elastic liposomes

membrane, which are adaptable to ambient stress allow the ultra deformable liposomes to

change its membrane composition locally and reversibly when it is pressed against or

attracted into a narrow pore. The elastic liposomes components that sustain strong

membrane deformation preferentially accumulate, while the less adaptable molecules are

diluted at sites of great stress. This dramatically lowers the energetic cost of membrane

deformation and permits the resulting high flexible particles first to enter and then to pass

through the pores rapidly and efficiently. This behavior is not limited to one type of pore

and has been observed in natural barrier such as in intact skin (Cevc, 1993a; Paul et al.,

1995a).

Natural transdermal water concentration gradient consequently drives high

number of the specially designed lipid vesicles across the hydrophobic outer skin layers.

This does not pertain to all lipid vesicles. The available transdermal osmotic pressure

difference is too low to push the standard lipid vesicles (liposomes) through an intact

mammalian stratum corneum. Dermally, applied lipid vesicles can only penetrate into

rather than across this region. The reason for this is the prohibitively high cost of the

standard liposome deformation. In order to increase the efficacy of vesicle penetration

through the skin it is therefore necessary to minimize this cost for each given vesicle

type. It had been by adjusting the lipid bilayer composition until the maximum of the

tolerable vesicles surface flexibility was achieved. Such an optimization yields elastic

liposomes (Cevc and Blume, 1992).

2.5.8.1 Salient features and advantages of elastic liposomes

1. Elastic liposomes possess an infrastructure consisting of hydrophobic and

hydrophilic moieties together and as a result can accommodate drug molecules with

a wide range of solubility.

2. Elastic liposomes can deform and pass through narrow constriction (from 5 to 10

times less than their own diameter) without measurable loss. This high

deformability gives better penetration of intact vesicles.

Chapter 2 - Review of literature

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3. Elastic liposomes can act as a carrier for low as well as high molecular weight drugs

e.g. analgesics, anesthetics, corticosteroids, sex hormones, anti-cancer agents,

insulin, gap junction protein and albumin.

4. Elastic liposomes are biocompatible and biodegradable, as they are made from

natural phospholipids similar to liposomes.

5. Elastic liposomes have high entrapment efficiency, in case of lipophilic drug near to

90%.

6. Elastic liposomes protect the encapsulated drug from metabolic degradation.

7. Elastic liposomes act as depot releasing their contents slowly and gradually.

8. Elastic liposomes can be used for both systemic as well as topical delivery of drug.

9. Easy to scale up as procedure is simple, do not involve lengthy procedure and

unnecessary use of pharmaceutically unacceptable additives.

2.5.8.2 Elastic liposomes vs other carrier systems

At first glance, elastic liposomes appear to be remotely related to lipid bilayer

vesicle liposomes. However in functional terms, elastic liposomes differ vastly from

commonly used liposomes in that they are much more flexible and adaptable. The

extremely high flexibility of their membrane permits elastic liposomes to squeeze

themselves even through pores much smaller than their own diameter. This is due to high

flexibility of the elastic liposomal membrane and is achieved by judiciously combining at

least two lipophilic/amphiphilic components (phospholipids plus biosurfactant) with

sufficiently different packing characteristics into a single bilayer. The resulting high

aggregate deformability permits elastic liposomes to penetrate the skin spontaneously.

This tendency is supported by the high elastic liposomal surface hydrophilicity that

enforces the search for surrounding of high water activity. It is almost certain that the

high penetration potential of the elastic liposomes is not primarily a consequence of

stratum corneum fluidization by the surfactant because micellar suspension contains

much more detergent than elastic liposomes (PC/Sodium cholate 65/35 w/w %,

respectively). Thus if the penetration enhancement via the solublization of the skin lipids

was the reason for the superior penetration capability of elastic liposomes, one would

Chapter 2 - Review of literature

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expect an even better penetration performance of the micelles. In contrast to this postulate

the higher detergent concentration in the mixed micelles does not improve the efficacy of

material transport into the skin. On the contrary mixed micelles stay confined to the top

most part of the stratum corneum even when they are applied non-occlusively (Cevc et

al., 1995). The reason for this is that mixed micelles are much less sensitive to the trans

epidermal water activity gradient than elastic liposomes. Elastic liposomes differ in at

least two basic features from the mixed micelles; first elastic liposome is normally by one

to two orders of magnitude (in size) greater than standard lipid micelles. Secondly, and

more importantly each vesicular elastic liposome contains a water filled core whereas

micelles are just simple fatty droplets. Elastic liposomes thus carry water as well as fat-

soluble agent in comparison to micelles that can only incorporate lipoidal substances

(Schatzlein and Cevc, 1995; Planas et al., 1992).

To differentiate the penetration ability of all these carrier systems Cevc et al.

(1996) proposed the distribution profiles of fluorescently labeled mixed lipid micelles,

liposomes and elastic liposomes as measured by the Confocal Laser Scanning

Microscopy (CLSM) in the intact murine skin. In all these vesicles the highly deformable

elastic liposomes transverse the stratum corneum and enter into the viable epidermis in

significant quantity.

Chapman and Walsh, (1990) also showed that the former two types of aggregates

are confined to the outer half of the horny layer where the cellular packing and

intercellular seals are already compromised by the desquamation process. Pure lipid

vesicles or micelles seem to have access to the low-resistance pathway only and thus very

seldom reach the lower stratum corneum or even get into the viable parts of the skin in

significant quantities.

Jain et al. (2006) Elastic liposomes penetrate the skin to depend greatly on the

high vesicle deformability and act as a drug reservoir to continuously transport drug

through the skin.

Chapter 2 - Review of literature

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In contrast to this, the much more deformable vesicles can use intra-as well as

intercluster pathway by virtue of their capability to squeeze themselves into the smallest

pores. Such lipid aggregates consequently are found in the stratum corneum as well as in

the viable skin and even bring their associated material deep into the body (Cevc et al.,

1996).

2.5.8.3 Basic principle behind development of elastic liposomes

Elastic liposomes when applied under suitable condition show high permeation

ability across the skin (Gompper and Kroll, 1995). The reason for this high flux rate is

naturally occurring transdermal osmotic gradient i.e. another much more prominent

gradient is available across the skin. This osmotic gradient is developed due to the skin

penetration barrier which prevents water loss through the skin and maintains a water

activity difference in the viable part of the epidermis (75% water content) and nearly

completely dry stratum corneum near to the skin surface (15% water content) (Rand and

Parsegian, 1990). This gradient is very stable because ambient air is perfect sink for the

water molecules even when the transdermal water loss is unphysiologically high. All

polar lipids attract some water. This is due to the energetically favorable interaction

between the hydrophilic lipid residues and their proximal water. Most lipid bilayers thus

spontaneously resist an induced dehydration (Warner and Lilly, 1994). Consequently all

lipid vesicles made from the polar lipid vesicles move from the rather dry location to the

sites with a sufficiently high water concentration. So when a lipid suspension (elastic

liposomes) is placed on the skin surface, elastic liposomes are partly dehydrated by the

water evaporation loss and then the lipid vesicles feel this osmotic gradient and try to

escape complete drying by moving along this gradient. They can only achieve this if they

are sufficiently deformable to pass through the narrow pores in the skin because elastic

liposomes composed of surfactant have more suitable rheologic and hydration properties

that are responsible for their greater deformability. Less deformable vesicles including

standard liposomes are confined to the skin surface where they dehydrate completely fuse

and hence they have less penetration capability than elastic liposomes. Elastic liposomes

Chapter 2 - Review of literature

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are optimized in this respect and thus attain maximum flexibility and can take full

advantages of the transepidermal osmotic gradient (water concentration gradient)

(Honeywell-Ngugen et al., 2002).

2.5.8.4 Safety considerations

Phospholipid suspensions comprising liposomes were reported to be harmless and

non-irritating to the skin after repeated epicutaneous administration; they may even have

additional advantageous cosmetic effect. Macroscopic observations made with elastic

liposomes indicate in the same direction. Such ultra deformable vesicles tested on the

skin in a microscopic toxicity assay revealed no difference between the saline as a

negative control and various elastic liposomal formulations (Van den Bergh et al., 1999).

From the point of view of systemic toxicity similarly favorable situation is

expected. Main component of elastic liposomes is typically Soya Phosphatidylcholine of

greater than 95% purity, which is generally regarded as safe because it is already used as

emulsifier in microemulsion for the parenteral nutrition and also used in injectable drug

formulation. In light of these data one can expect the elastic liposomal product to be very

safe from the carrier point of view (Jain et al., 2005b). Table 2.11 summarizes the

different studies reported by using elastic liposomes as carrier for drug delivery.

Chapter 2 - Review of literature

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Table 2.11:Studies reported for application of elastic liposomes as carriers.

Drug Name System Administration

route

Finding Reference

Interleukin-2

and interferon-α

Elastic liposomes Transdermal Avoids injection

Hofer et al.

(2000)

Corticosteroids Elastic liposomes Dermal Sustained release of

drug

Superior biological

activity

Jain et al. (2003a)

Norgestrel Elastic liposomes Transdermal Provides effective

contraception

Better stability

Easy to scale up

Jain et al.

(2003b);

Jain et al.

(2005b)

Diclofenac Elastic lipoosmes Dermal Improved analgesic

effect

Non invasive treatment

of local pain.

Jain et al.

(2005b)

Zidovudine

(AZT)

Elastic liposomes Dermal Sustained and targeted

delivery of drug

Jain et al. (2006)

Melatonin Elastic liposomes Transdermal Sustained and targeted

delivery of drug

Dubey et al.

(2006)

Propranolol

Hydrochloride

Elastic liposomes Transdermal Avoidance of first pass

metabolism

Better therapeutic

efficiency

Mishra et al.

(2007)

Zidovudine PEGylated elastic

liposomal

formulation

Transdermal Higher accumulation of

drug in lymphoid tissue

Jain et al.

(2008)

Docetaxel Elastic liposomes Transdermal Enhanced transdermal

flux

Qiu et al.

(2008)

Rizatriptan Elastic liposomes Transdermal Improved transdermal

permeability

Reduced side effects

Garg et al., 2008

Colchicine Elastic liposomes Transdemal Improve therapeutic

activity

Singh et al.

(2009)

Valsartan Elastic liposomes Transdermal Better skin permeation

Imroved anti-

hypertensive activity

Ahad et al.

(2011)

Benzocaine

(BZC)

Drug-in

cyclodextrin in

elastic liposomes

Dermal Enhancement of

intensity and duration of

anaesthetic effect

Maestrelli et al. (2010)

Ketorolac Elastic liposomes Transdermal Deep penetration of drug

into skin

Nava et al.

(2011)

Diclofenac

sodium

Elastic liposomes Transepidermal Enhanced

transepidermal flux

Sustained release

Zaafarany et al. (2010)

Isotrietion Drug in cyclo

dextrnin elastic

liposomes

Dermal Sustain release Kaur and

Jain, (2012)

Chapter 2 - Review of literature

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2.6 Conclusions

Novel drug delivery systems of anti-cancer drugs will alter the pharmacokinetic

properties of drug accompanied by elimination or reduction of non specific toxicities

typically associated with chemotherapy. They will provide versatile and straight forward

approach for improving the physiological and pharmacological responses of the drug and

overcome the problem of drug solubility. With use of novel drug delivery systems it is

possible to reduce the dose of drug and at the same time cost of cancer chemotherapy.

Novel approaches to cancer treatment not only supplement the conventional

chemotherapy and radiotherapy but also prevent damage to normal tissues and prevent

drug resistance.