Tunaidi AnsariMay 3, 2008

Biodegradable Long-Circulating Polymeric NanospheresR. Gref, Y. Minamitake, Y. Peracchia,

V. Trubetskoy, V. Torchilin, and R. Langer

There are six specific features that form an efficient carrier, which can continuously deliverdrugs intravenously. These include: 1) encapsulated agents of relatively high weight fractioncompared to the rest of the carrier system, 2) efficient incorporation of the agents from the firststep to the final carrier, 3) competence in freeze-drying and subsequent thawing, 4)biodegradability, 5) small size, and 6) ability to stay in the blood stream for relatively longerperiods of time [1].

Gref et al developed degradable polymeric nanospheresthat exhibit the previously listed features of an efficientcarrier. Biodegradable materials known to be harmlessin the human body, such as poly(lactic-co-glycolic acid)(PLGA) (shown in Figure 1), polycaprolactone (PCL),and their copolymers, were used as the core of theseparticles [2,4,6,7]. In order to form adequate coating,polyethylene glycol (PEG) was covalently bonded to thenanosphere core. This was accomplished by diblockcopolymers, PEG-R, and R being one of the listedbiodegradable materials. Since these diblock polymersare amphiphilic, they were used to obtain a phase-separated structure.

The nanospheres were constructed by first dissolving PEG-R in an organic solvent. Subsequentvortexing and sonication formed an emulsion in an aqueous phase. This is because PEG is

hydrophilic and R is lipophilic [5]. Afterwards,evaporation of the solvent yielded a solidifiednanosphere core. These were collected throughcentrifugation and then lyophilized for easyredispersion in aqueous solutions.

Quasi-elastic light scattering (QELS) verifiedthat aggregation did not occur. Atomic forcemicroscopy (AFM) was utilized to confirm thenanospheres’ spherical shape, reporting a meandiameter of 90 and 150 nm. X-ray photoelectronspectroscopy (XPS) established that PEG wasconcentrated mainly within 5 nm of thenanospheres’ outer layer, and only a nominalamount of PEG was detached.

In Figure 2, (A) and (B) show images of

Figure 2: Images of nanospheres and relatedplots.

Figure 1: Illustration of the chemicalstructure of PLGA [3].

nanospheres and respective lengths taken from the AFM, where (A) is PLGA and (B) is PEG-PLGA. (C) depicts the QELS results and displays diameters of the PEG-PLGA nanospheres.Finally, (D) displays the XPS analysis, where trace 1 corresponds to PLGA nanospheres, trace 2corresponds to PEG-PLGA nanospheres, and trace 3 corresponds to PEG alone.

PEG-coated and non-coated particles were injected into mice in orderto determine their respective effectiveness. As shown in Figure 3 (A),as the molecular weight of PEG increases, there is a correspondingincrease in blood circulation time. This is due to the increased thicknessof the PEG coating, which lessens the effect of opsonization. Inaddition, Figure 3 (B) proves that within five minutes, 66% of the non-coated particles were removed by the liver, while fewer than 30% ofthe PEG-coated nanospheres met the same fate only after five hours.Gamma scintigraphy proved that non-coated nanospheres were presentonly in the liver and spleen, while large amounts of PEG-coatedparticles were discovered in the blood pool, mainly the heart and lungs[18].

Next, lidocaine, a model drug, wasencapsulated into the PEG-coatednanospheres and achieved high drugloadings and entrapment efficiencies.Lidocaine was continually released invitro for over 14 hours. It was established

that the higher the particle drug content, the slower the release. Thistrend can be seen in Figure 4 (A).

This experiment by Gref et al presents a novel development ofintravenously administered carriers with adequate blood circulationtimes. It is improves upon previously attempted projects such asalbumin or galactose microspheres, which are frequently used inclinical studies for imaging [19,20,21]. However, the downfall ofthese microspheres is their rapid clearance from the blood within 20seconds, which prevents subsequent usage in various imagingapplications. Other innovations also challenged the rapid clearancerate frontier. Progress has been made on the micro-particle scale byappending poloxamer or polysorbate onto nondegradable polystyrene oparticles. Another alternative that has been performed was the formatiocarriers containing glycolipids, albumin, or derivatives of PEG [22-30]further advancements into this field on the nano-particle scale and attemwith previously performed experiments.

When forming their nanosphere cores, they relied on the principle of adcompositions and molecular weights of polymers in order to preciselyof the core and the release kinetics of the encapsulated agents [8]. Opsodetection present barriers to long term blood circulation [17]. As such,

Figure 3: Data onmice, which were

injected with varyingmolecular weights of

PEG-PLGA.

Figure 2: Results pertainingto lidocaine drug release.

r polymethylmethacrylaten of liposomes and other. Gref et al hoped to makepted to solve the issues

justing chemicalcontrol degradation timesnization and macrophagePEG was selected as a

component of the nanosphere coating. Since the diblock copolymers that were used consisted ofethylene glycol groups bonded together and attached to repeated hydrophobic monomers, Gref etal were able to utilize the different solubilities of the PEG and R to acquire the phase-separatedorganization. This was used in the emulsion process of the nanosphere formulations.

The lidocaine-loaded nanospheres, which exhibit the inverse relationship between drug contentand release speed, may be accounted for by drug crystallization within the nanospheres.Referring to Figure 4 (B), calorimetric and x-ray diffraction have shown that with low content,the drug exists as a dispersion inside the core and with high content, phase separation causes aportion of lidocaine to be crystallized [14,15]. Thus, in order to control timing of drug-release,several factors are involved. These include: manipulation of the diblock polymers’ chemicalcomposition, effective drug loading, and optimal nanosphere particle sizes [9].

Gref et al’s innovation can be applied in multiple ways. Further research and experiments willinevitably allow these biodegradable long-circulating polymeric nanospheres to be fruitful inareas such as drug delivery, medical imaging, and gene therapy. Specific applications includeattaching a suitable protein to the surface of the nanospheres, which may possibly permitendocytosis of DNA-containing particles, or even attaching antibodies to PEG, and thus forminghighly specific immune defense systems [10,11,12].

Nevertheless, there may be areas of improvement regarding this particular study. Researchingother types of particle coating most effective for their functions or delivery target may be evenmore useful in future applications. For example, amylose serves as an effective coating forcarriers targeting the colon [13]. Alternatively, further investigation into the unloading propertiesof agent release may be useful for specific drug delivery and the timing of their resulting effects.This may be directly applied to patients who need to regulate insulin levels as a result of diabetesmellitus. Since diabetes mellitus is a chronic disease, administered medication needs immediateeffect; altering timing and release of drugs from the nanospheres would therefore serve to bettermonitor and suppress the symptoms of the disease [16].

A very practical next step may encompass evaluating the different environmental factors, such aspH, temperature, and solvents, and their effects on the nanoparticle PEG coating. Regulatingthese factors is useful for determining the speed of degradability and subsequently the release ofagents. Controlling the endurance of the protective layer will be a milestone achievement instudying the encapsulation properties of the nanospheres. Additionally, further research into thedifferences between the various materials used in the R segment of the PEG-R complex mayhelp to fine tune the experimental results. Perhaps, even the integration of triblock copolymersonto the nanospheres can be performed and studied for comparison with the currently useddiblock copolymers.

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