SILK-BASED DELIVERY SYSTEMS OF BIOACTIVE MOLECULES

Presented by: Sk.Asma Shaheda

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CONTENTS Introduction Sources of silk proteins 1.Natural -Silk worm fibroin -Spider silk fibroin 2. Recombinant -Silk worm variants -Spider silk variants Advantages of silk proteins Preparation of Sericin free silk solution Biosynthesis of recombinant spider silk proteins - Design, construction, and cloning of the genes,

and - Expression and purification of the protein

polymers.

Applications of Silk worm silk protein for drug delivery with various examples a. Scaffolds b. Silk films c. Nanofibres d. Microspheres e. Nanoparticles f. Microneedles g. Hydrogels h. Coatings Applications of recombinant Spider silk for drug and gene delivery with various examples a. Reconstituted spider silk as microspheres b. Spider silk-polycation block copolymers c. spider silk-polycation functional peptide multiblock copolymers

Release of drug from silk matrix and fate of silk fibroin Conclusion References

INTRODUCTION:

Silk based delivery systems deals with the use of silk protein as a polymer for various drug delivery systems.

Silks are biodegradable, biocompatible, self-assembling proteins that can be tailored via genetic engineering to contain specific chemical features, offering its utility for drug and gene delivery.

This topic focuses on the biosynthesis of silk-based polymer systems and related silk protein drug delivery.

SOURCES OF SILK PROTEINS:

1.Natural silk proteins: Produced by a variety of insects and spiders. Biodegradable and possess high mechanical properties. Functions include development(cocoons), prey capture(spider webs), to safety lines(Spider dragline). Have their applications in biomedical suture, biomaterial culture and tissue engineering. a. Silk worm fibroin: It has its own applications in biomedical suturing for

decades and in textile production for clothing for centuries.Structure: Silk is a continuos strand of two filaments cemented

together forming the cocoon of silk worm. Silk filament –double strand of fibroin-held by Silk sericin. Silk fibroin -glycoprotein and composed of 2 equimolar

protein subunits, -light and heavy chain fibroins linked by disulphide bonds.Best example of β-pleated structure.

Beta pleated structure of the silk

Amino acid composition of the fibroin

b. Spider silk fibroin:Primary structure is its amino acid sequence with highly repetitive glycine and alanine blocks, which are referred to as block copolymers. Protein composition in the primary structure

Large Hydrophobic blocks with Small hydrophilic blocks with more highly conserved sequences consisting complex sequences that consist Of short side-chain amino acids such as aminoacids with bulkier side-chains Glycine and alanine. and charged aminoacids.

Show α-helices in the solutionAnd β-sheet structures in the assembled form

Thus a primary structure possess a amphiphilic composition as that of surfactants and biological membranes. During secretion from the spinning duct, repetitive sequences undergo intra and inter-molecular interactions resulting in the formation of secondary, tertiary, quaternary structure.

a. Secretion of silk thread from the spinning duct b. Structure of spider silk fibroin

Non-protein composition of spider silk: Compound(s) Uses MechanismSugars, lipids, ions, and pigments

Act as protection layer in the final fibre

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Pyrollidine Keeps thread moist

Due to its hygroscopic properties

Potassium hydrogen phosphate

Make the silk acidic and protect from fungi and bacteria that digest the protein

Releases protons in aqueous solution, resulting in pH-4

Potassium nitrate Prevent from denaturing in the acidic medium

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DIFFERENCES BETWEEN SILKWORM AND SPIDER SILK :-

SILK WORM SILK

SPIDER SILK

Molecular level

Large amount of sericin is present

Sericin is absent

Proteins responsible for fibrillar structures

Called as fibroins and contains light and heavy fibroins

Called as fibroins or spidroins specifically and contains light and heavy counter-parts

Mechanical properties

Weaker and less extensible

Stronger with high extensible properties

Spinning conditions

Either strong or elastic

Both strong and elastic

2.Recombinant silk proteins:a. Silk worm variants: Silk like repeats of (GAGAGS), elastic block copolymers, Silk-

elastin-like proteins (SELP)Results: Enhanced gene expression was reported in target cells up to 10

fold, when compared to viral injection without the SELP. With insertion of partial collagen and fibronectin sequences,

cell-adhesive ability was increased. Films made from recombinant silk proteins had six-fold higher

activity than original silk fibroin.b. Spider variants: Spider silk sequence was modified to contain methionines

adjacent to polyalanine sequence, controlled self assembly of beta-sheet structures in silk.

Modified spider silk, which was 15mer of [SGRGGLGGQGAGAAAAAGGAGQGGYGGLGSQGT] derived from the spidroin was bioengineered to include arginyl-glycyl-aspartic acid(RGD) cell-binding domains to enhance cell adhesion.

Also, hydrophilic [SQGGYGGLGSQGSGRGGLGGQT] and hydrophobic blocks [SGAGAAAAAGGAGT] were combined and cloned with different hydrophilic and hydrophobic blocks ratios.

Advantages of silk proteins as biomaterials for drug delivery:Delivery of Bioactive molecules and drugs in slow, sustained, controlled release formats. Biodegradable, biocompatible, and mechanically durable. Processed under ambient aqueous conditions to avoid loss of bioactivity of drugs to be delivered. Less inflammatory than other common biodegradable polymers such as poly(lactide) and collagen. Proccessability into films, hydrogels, nano-fibres, and three-dimensional scaffolds. Degradation rate can be adjusted by controlling the crystalline state(β-sheet) during processing, in order to regulate release profile of bioactive molecules. Spider silk-based block copolymers have been designed via genetic engineering and used for the delivery of bioactive molecules, like genes and drugs. Selective delivery to target cells.Eg: Silk proteins containing tumor-homing peptides as nano-particles ---targeting tumor cells.

Preparation of sericin free silk solution:Sericin protein is a potential allergen causing allergic and cytotoxic reactions. Hence removal of sericin is necessary.It includes the following steps as shown in the diagram:

Biosynthesis of recombinant silk-like polymers:

APPLICATIONS OF SILK WORM SILK PROTEIN FOR DRUG DELIVERY WITH VARIOUS EXAMPLES :a. Scaffolds: Scaffolds have been prepared by using Salt leaching method as shown below

b. Silk films: Prepared by cast or layer-by-layer deposition with various concentrations.

c. Nanofibres: They can be prepared by electrospinning.

d. Microspheres: They were processed using spray drying and lipid vesicles.With spray drying microspheres of 100µm size was produced which is sub-optimal for drug delivery.Hence, lipid vesicle method is followed.

e. Microneedles: Silk fibroin based microneedles were developed for delivery of drugs and other compounds directly to tissue in a controlled manner.Chemical properties of the embedded substances is maintained.Water vapour annealing and various temperature exposures provided control overthe diffusivity of silk microneedles and drug release kinetics.

a. Process of development of silk microneedles b. Implantation of patch of microneedles loaded with TTC

f. Nanoparticles: Silk based nanoparticles from silk fibroin solutions were stable, spherical, negatively charged, 150-170nm in average diameter and showed no toxicity.

g. Hydrogels:Hydrogels of silk fibroin are formed via sol-gel transitions by sonication, vortexing, or the presence of acid and /or ions.

h. Coatings:Silk fibroin solution was applied as coating over the delivery systems like microspheres, nano-particles or directly on the drug surface in order to get a sustained release of the drug. The thickness of one layer was reported to be around 10nm when deposited from a 1mg/ml silk aqueous solution. Release from these coatings can be controlled via layer thickness, number of layers and secondary structure of the fibroin layer.

Loaded bioactive molecule

Type of delivery Effect produced

Bone morphogenetic protein-2(BMP-2)

Scaffolds •Sustained release•Induced Human bone marrow stromal cells to under go osteogenic differentiation

nanofibres Supported high calcium deposition and enhanced transcription of bone specific markers.

BMP-2, BMP-9, BMP-14

Microspheres Slow release up to 14days.

BMP,RGD, Parathyroid harmone(PTH)

Silk films Differentiation of human bone marrow derived stem cells with silk films was induced by immobilised BMP-2

Horseradish peroxidase (HRP) enzyme

ScaffoldsMicrospheresSilk filmMicroneedles

Controlled and sustained release of enzyme over 10-15days without effecting its bioactivity

Various examples of drugs that have been loaded using silk fibroin:

Loaded bioactive molecule

Type of delivery

Effect produced

Adenosine Silk filmsImplants

Promote long term adenosine release from adenosine kinase deficient embryonic stem cells for over a period of 2weeks via slow degradation of silk and delivery of predetermined dose

Enzymes like Glucose oxidase, Lipase, HRP

Silk films •Stabilization of entrained molecules •Entrained molecules retained significant activity over 10months even when stored in 37°C

Curcumin Nano-particles Showed higher efficiency against breast cancer cells and have potential to treat in-vivo breast tumors by local, sustained, and long-term therapeutic delivery

Growth factors Nano-particlesMicrospheres in alginate gels

•Sustained release over 3weeks.•More efficient in delivering BMP-2 than insulin-like growth factors

Tetracyclines Microneedles Inhibited local infection of Staphylococcus aureus

Coatings:Eg:- 1. -Poly(lactide-co-glycolic acid) (PLGA) microspheres -Alginate microspheres Coated with silk fibroin solution formed mechanical shells as well as diffusion barrier to the encapsulated drugs.

2.Nano layer coating on small molecule drugs and therapeutically relevant proteins like rhodamine-B and Azoalbumin was achieved.

3.Multilayered silk-based coatings was given to evaluate vascular responses to heparin, paclicoxel, and clopidiogrel-----Paclitaxel, clopidiogrel inhibited smooth muscle cell proliferation and retarded endothelial cell proliferation.-----Silk multilayers of Heparin promoted human aortic endothelial cell proliferation while inhibited human coronary artery smooth cell proliferation which is a desired outcome in restenosis.

4.Solid adenosine powder coated with silk fibroin-----showed Local and sustained delivery-----Increase in either coating thickness or crystallinity Delayed adenosine burst Decreased daily release rate of adenosine Resulting in increased duration of action

Applications of recombinant spider silk to drug delivery: a. Reconstituted spider silk: The reconstituted dragline silk proteins

have been used to prepare microcapsules for drug delivery. Microspheres may offer

potential for the development of targeted drug delivery systems. Various other delivery systems into which the spider silks can be formulated have been shown in the following image.

b. Spider silk-polycation block copolymers:Poly(L-lysine) is a cationic polymer that interacts with DNA through electrostatic interactions to assemble into polyelectrolyte complexes, Which is used as an alternative to recombinant viruses for the delivery of pDNA into cells. But it showed low transfection efficiency.

A. Schematic representation of silk-based pDNA complexes and silk films containing the complexes. Silk-based polyioncomplexes are formed between negatively charged pDNA and positively charged polylysine sequence of silk-polylysine block copolymer. Silk-based polyioncomplexes amd films to contain the complexes are prepared for pDNA deliveryB. pDNA complexes of the recombinant silk(yellow spots).

c. Spider silk-polycation-functional peptide multiblock copolymers:Silk based block copolymers are potentially useful candidates for nonviral genevector because various functional peptides such as cell binding motifs (RGD), cell penetrating peptides(cPP), signal peptides of virus, and or tumor-homing peptides can be added as ligands through recombinant DNA techniques.

Model of receptor mediated transfection via silk-based cationic block copolymers with ligands or functional peptides. (a) Formation of ion complexes between gene(s) and silk-polylysine block copolymers. (b) Binding of the complex to the cell via specific receptors or membrane proteins such as integrins. (c) Internalization via endocytosis and degradation of polymers in lysosomes. (d) trafficking of genes to the nucleus to initiate gene expression after the degradation of the complex. (e) Binding of adenovirus vector to the cell via the coxsackievirus and adenovirus receptor(CAR). (f) Internalization via the receptor-mediated endocytosis, involving interactions between integrins and RGDs in the adenoviral penton capsid protein. (g) Dismantling of capsid and acidification endosome, and subsequent docking at nuclear pore complexes and passage of DNA through nuclear pores via interaction of naked capsid with microtubules and dynein motors.

Release of drug from silk matrix and fate of silk fibroin:Drug is released in a controlled manner for a long period of time.Release kinetics depends on –Adjusting crystallinity, concentration and structure of silk fibroin, design of delivery system as well of molecular weight and structure of embedded agents.

Eg:

FATE: Biosdegradation by proteolytic enzymes such as chymotrypsin, actinase, carboxylase which involves two steps.Adsorption of silk biomaterial by different enzymesDigestion by enzymesFinal wastes are easily absorbed invivo

ConclusionSilk-based biomaterials are used to deliver bioactive molecules, such as small drugs, proteins, genes.

They show remarkable mechanical properties, versatile processing in an aqueous environment, biocompatibility, and controlled degradation suggest silks as attractive biomaterials for controlled and sustained release, stabilization and delivery of bioactive molecules.

Silk solutions can be morphed into a variety of biomaterial formats, including films, 3D porous scaffolds, hydrogels, micro- and nano-spheres, nanofibres and coatings.

Targeted delivery can be achieved

Hybrid or composite silk-based materials containing other biopolymers, have not been extensively studied, yet should provide applicable mechanical, thermal, and biological properties for not only drug/gene delivery but also for tissue engineering, medical imaging, and regenerative medicine.

REFERENCES:1.http//www.ncbi.nlm.nih.gov/pmc/articles/PMC2658765/

2.http://now.tufts.edu/news-release/silk microneedles-deliver-drugs-

3. http://en.wikipedia.org/wiki/Silk

4. Journal of control release,vol-150,issue2,10 march2011,pg no:128-1415.

5. International journal of molecular science,march-31,2009,pg no:1514-1524

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