Preparation of Biodegradable,Surface Engineered PLGA Nanospheres with Enhanced Lymphatic Drainage and Lymph Node Uptake

Purpose. Nanospheres can be utilised for the targeting of drugs and diagnostic agents to the regional lymph nodes. The surface modification of model polystyrene, (PS), and poly(lactide-co-glycolide),(PLGA), nanospheres by poly(lactide)-poly(ethylene glycol), (PLA:PEG), copolymers has been assessed by in vitro characterisation and in vivobiodistribution studies following subcutaneous administration of the nanospheres to the rat. Methods. Three PLA: PEG copolymers were investigated, with PEG chain lengths of 750, 2000 and 5000 Da. The PLA:PEG copolymers were either coated onto the surface of PS and PLGA nanospheres or used as a co-precipitate in the formation of PLGA-PLA:PEG nanospheres. Coating of the nanospheres was confirmed by an increase in their particle size and a corresponding decrease in the surface potential. The kinetics of injection site drainage and lymph node retention was determined over a 24 hour time course for naked, coated and co-precipitated nanosphere systems. Results. Dependent on the surface characteristics, the distribution of the nanospheres can be significantly modified and the lymph node localisation dramatically enhanced by coating their surfaces with PLA:PEG copolymers or by producing co-precipitate nanospheres of PLGA and PLA:PEG. Conclusions. A fully biodegradable nanosphere system has been developed with excellent lymph node targeting characteristics.     

Surface Modification of Poly(lactide-co-glycolide) Nanospheres by Biodegradable Poly(lactide)-Poly(ethylene glycol) Copolymers

The modification of surface properties of biodegradable poly(lactide-co-glycolide) (PLGA) and model polystyrene nanospheres by poly(lactide)-poly(ethlene glycol) (PLA:PEG) copolymers has been assessed using a range of in vitro characterization methods followed by in vivo studies of the nanospheres biodistribution after intravenous injection into rats. Coating polymers with PLA:PEG ratio of 2:5 and 3:4 (PEG chains of 5000 and 2000 Da, respectively) were studied. The results reveal the formation of a PLA: PEG coating layer on the particle surface resulting in an increase in the surface hydrophilicity and decrease in the surface charge of the nanospheres. The effects of addition of electrolyte and changes in pH on stability of the nanosphere dispersions confirm that uncoated particles are electrostatically stabilized, while in the presence of the copolymers, steric repulsions are responsible for the stability. The PLA:PEG coating also prevented albumin adsorption onto the colloid surface. The evidence that this effect was observed for the PLA:PEG 3:4 coated nanospheres may indicate that a poly(ethylene glycol) chain of 2000 Da can provide an effective repulsive barrier to albumin adsorption. The in vivo results reveal that coating of PLGA nanospheres with PLA:PEG copolymers can alter the biodistribution in comparison to uncoated PLGA nanospheres. Coating of the model polystyrene nanospheres with PLA:PEG copolymers resulted in an initial high circulation level, but after 3 hours the organ deposition data showed values similar to uncoated polystyrene spheres. The difference in the biological behaviour of coated PLGA and polystyrene nanospheres may suggest a different stability of the adsorbed layers on these two systems. A similar biodistribution pattern of PLA:PEG 3:4 to PEG 2:5 coated particles may indicate that poly(ethylene glycol) chains in the range of 2000 to 5000 can produce a comparable effect on in vivo behaviour.     

Polylactide-poly(ethylene Glycol) Micellar-like Particles as Potential Drug Carriers: Production,Colloidal Properties and Biological Performance

The micellar-like particle systems produced from poly-D,L-lactide-poly(ethylene glycol) (PLA-PEG) copolymers have been assessed using a range of physicochemical characterisation methods, followed by in vivo studies of their biodistribution after intravenous administration to the rat. The size of the PEG chain was kept constant at 5 or 2 kDa, while the PLA size increased within a series from 2 to 25 kDa. The results obtained reveal, that in an aqueous medium the copolymers assembled into micellar-like structures, with the PLA segments forming the core and the PEG segments the surrounding corona. The size of the PLA segments dominated the process of assembly of the molecules and the characteristics of the resultant micellar-like particles. The PLA-PEG micellar particles were found to be less dynamic than those obtained from conventional surfactants. Particles formed from the lower molecular weight PLA polymers allowed a level of chain mobility while the cores of the micellar particles formed from higher molecular weight PLA appeared to be solid-like in nature. The size of the micellar particles was dependent on the copolymer molecular weight and the z-average diameter increased from 25 to 76 nm as the molecular weight of the PLA moiety increased. This provides an ability to control the particle size by adjusting the molecular weight of the PLA moiety. Following intravenous administration to the rat model, micellar-like particles smaller than approximately 70 nm accumulated in the liver, despite the fact that the PEG corona provided an effective steric stabilization effect. Micellar-like particles with a diameter of more than approximately 70 nm exhibited prolonged systemic circulation and reduced liver uptake, although the steric stabilisation of these particles was shown to be less effective. These findings agree with recent observations from other research groups; that indicate a possibility that very small particulates can pass through the sinusoidal fenestrations in the liver and gain access to the parenchymal cells of the liver.     

The Structure of PEG-Modified Poly(Ethylene Imines) Influences Biodistribution and Pharmacokinetics of Their Complexes with NF-κB Decoy in Mice

Purpose. To study the relationship between structure of poly(ethylene imine-co-ethylene glycol), PEI-PEG, copolymers and physicochemical properties as well as in vivo behavior of their complexes with NF-B decoy. Methods. A variety of copolymers of PEG grafted onto PEI as well as PEI grafted onto PEG were synthesized and their complexes with a double stranded 20mer oligonucleotide were examined regarding size, surface charge, biodistribution and pharmacokinetics. Results. Polyplexes of copolymers were smaller compared to polyplexes formed by non-PEGylated PEI 25 kDa (58 - 334 nm vs. 437 nm for a nitrogen/phosphate ratio of 3.5 and 85 - 308 nm vs. 408 nm for N/P 6.0) and showed reduced zeta potential (–2.5 - 6.4 mV vs. 14.5 mV for N/P 6.0). IV injection into mice revealed liver (35-76 % of injected dose), kidney (3 - 22 %) and spleen (2 - 16 %) to be the main target organs for all injected complexes. Complexes formed by copolymers with few PEG blocks of higher molecular weight (5 kDa and 20 kDa) grafted onto PEI 25 kDa did not show different blood levels from PEI 25 kDa. In contrast, a copolymer with more short PEG blocks (550 Da) grafted onto PEI showed elevated blood levels with an increase in AUC of 62 %. Conclusions. A sufficiently high density of PEG molecules is necessary to effectively prevent opsonization and thereby rapid clearance from blood stream.     

Stealth PLA-PEG Nanoparticles as Protein Carriers for Nasal Administration

Purpose. The aim of the study was to encapsulate a model protein antigen, tetanus toxoid (TT), within hydrophobic (PLA) and surface hydrophilic (PLA-PEG) nanoparticles and to evaluate the potential of these colloidal carriers for the transport of proteins through the nasal mucosa. Methods. TT-loaded nanoparticles, prepared by a modified water-in-oil-in-water solvent evaporation technique, were characterized in their size, zeta potential and hydrophobicity. Nanoparticles were also assayed in vitro for their ability to deliver active antigen for extended periods of time. Finally, ¹²⁵I-TT-loaded nanoparticles were administered intranasally to rats and the amount of radioactivity recovered in the blood compartment, lymph nodes and other relevant tissues was monitored for up to 48 h. Results. PLA and PLA-PEG nanoparticles had a similar particle size (137-156 nm) and negative surface charge, but differed in their surface hydrophobicity: PLA were more hydrophobic than PLA-PEG nanoparticles. PLA-PEG nanoparticles, especially those containing gelatine as an stabilizer, provided extended delivery of the active protein. The transport of the radiolabeled protein through the rat nasal mucosa was highly affected by the surface properties of the nanoparticles: PLA-PEG nanoparticles led to a much greater penetration of TT into the blood circulation and the lymph nodes than PLA nanoparticles. Furthermore, after administration of ¹²⁵I-TT-loaded PLA-PEG nanoparticles, it was found that a high amount of radioactivity persisted in the blood compartment for at least 48 h. Conclusions. A novel nanoparticulate system has been developed with excellent characteristics for the transport of proteins through the nasal mucosa.     

Polyethylenglycol-co-poly-D,L-lactide copolymer based microspheres: Preparation,characterization and delivery of a model protein

Purpose: To prepare and characterize polyethylenglycol-co-poly-D,L-lactide (PEG-D,L-PLA) multiblock copolymer microspheres containing ovalbumin. Microsphere batches made of Poly-D,L-lactide (PLA) homopolymers were prepared in order to evaluate how the presence of PEG segments into PEG-D,L-PLA copolymer could affect the behaviour of microspheres as carrier of protein drugs.

Methods: The PEG-D,L-PLA and PLA microspheres, loaded with the model protein ovalbumin, were prepared using double emulsion solvent evaporation method. The effect of PEG segments in the microparticles matrix, on the morphology, size distribution, encapsulation efficiency and release behaviour was studied.

Results: According to the results, PEG-D,L-PLA microspheres were more hydrophilic than PLA microparticles and with lower glass transition temperature. The surface of PEG-D,L-PLA microspheres was not as smooth as that of PLA microparticles, the mean diameter of PEG-D,L-PLA microparticles was bigger than that of PLA microspheres. Protein release from the microspheres was affected by the morphological structure of PEG-D,L-PLA microspheres and properties of PEG-D,L-PLA copolymer. This study suggests that PEG-D,L-PLA multiblock copolymer may be used as carrier in protein delivery systems for different purposes.     

Stability and Release Performance of a Series of Pegylated Copolymeric Micelles

Purpose. The aim of this work is to evaluate the capability of a series of biocompatible amphiphilic copolymers as a nano-sized drug carrier. Methods. The influences of the type of lactone monomer, the feed molar ratios of lactone/PEG, and the molecular weight of PEG on the performance and release behavior of micelles are investigated. Results. These pegylated amphiphilic copolymers efficiently form micelles with a low CMC value in the range of 10^–6-10^–7 M. The average particle size of micelles is 100 nm. The phenomenon of increasing particle size as increasing the chain length of poly(lactone) block is observed. The different hydrophobicity, based on chemical structure of poly(lactone), accounts for different interaction strength between indomethacin and hydrophobic inner core, which further influences the drug loading in copolymeric micelles and their release character. In addition, the PCL/PEG/PCL micellar solutions maintain their sizes at 4°C for 8 weeks without occurring significant aggregation or dissociation. Conclusions. A series of biocompatible pegylated amphiphilic copolymers have been elucidated possessing micellization potential to form nano-sized micelles in an aqueous environment, which enable incorporate hydrophobic drug and regulate drug release.     

Defining the drug incorporation properties of PLA-PEG nanoparticles

The drug incorporation and physicochemical properties of PLA-PEG micellar like nanoparticles were examined in this study using a model water soluble drug, procaine hydrochloride. Procaine hydrochloride was incorporated into nanoparticles made from a series of PLA-PEG copolymers with a fixed PEG block (5 kDa) and a varying PLA segment (3-110 kDa). The diameter of the PLA-nanoparticles increased from 27.7 to 174.6 nm, with an increase in the PLA molecular weight. However, drug incorporation efficiency remained similar throughout the series. Incorporation of drug into the smaller PLA-PEG nanoparticles made from 3:5, 15:5 and 30:5 copolymers did not influence the particle size, while an increase was observed for the larger systems comprising 75:5 and 110:5 copolymers. An increase in drug content for PLA-PEG 30:5 nanoparticles was achieved by increasing the theoretical loading (quantity of initially present drug). The size of these nanoparticles remained unchanged with the increasing drug content, supporting the proposed micellar type structure of the PLA-PEG 30:5 nanoparticles. The morphology of these systems remained unchanged both at low and high theoretical drug loadings. Formulation variables, such as an increase in the aqueous phase pH, replacement with the base form of the drug and inclusion of lauric acid in the formulation did not improve the incorporation efficiency of drug into PLA-PEG 30:5 nanoparticles. While poly(aspartic acid) as a complexation agent did not improve the drug incorporation efficiency of procaine hydrochloride, it did so for another water soluble drug diminazene aceturate. This may be attributed to a stronger interaction of diminazene aceturate with poly(aspartic acid) relative to procaine hydrochloride, as confirmed by thermodynamic analysis of isothermal titration calorimetric data. The drug incorporation and physicochemical characterisation data obtained in this study may be relevant in optimising the drug incorporation and delivery properties of these potential drug targeting carriers.     

Enzymatic triggered release of an HIV-1 entry inhibitor from prostate specific antigen degradable microparticles

To achieve sustained release of 3-ethyl-4-(4-methylisoxazol-5-yl)-5-(methylthio) thiophene-2-carboxamide (BFB0261), a new potent osteogenic compound for treating bone disorders, we prepared film formulations containing BFB0261 and the following newly synthesized biodegradable polymers by a solvent casting technique: poly(D,L-lactic acid) (PLA), poly(D,L-lactic acid-co-glycolic acid) (PLGA), poly(D,L-lactic acid)-block-poly(ethylene glycol) (PLA-PEG), and poly(D,L-lactic acid-co-trimethylene carbonate) (PLA-TMC) polymers or copolymers. Powder X-ray diffractometry (PXRD), differential thermal analysis (DTA), scanning electron microscopy (SEM), and tensile testing were performed to examine the physicochemical properties of these films. Almost all the films exhibited a smooth and homogeneous surface, as observed by SEM. In addition, PXRD and DTA revealed that BFB0261 existed in an amorphous state in the films. The in vitro release of BFB0261 from PLA100 (M(w): 251 kDa), PLAPEG9604H (PLA/PEG ratio: 96:4; M(w): 181 kDa), PLAPEG8515H (PLA/PEG ratio: 85:15; M(w): 51.5 kDa), or PLAPEG8020 (PLA/PEG ratio: 80:20; M(w): 33.7 kDa) films followed zero-order kinetics with slow release up to 12 weeks following incubation. Although release of BFB0261 from PLA-TMC films followed first-order kinetics, sustained release of BFB0261 for 12 weeks was still observed for PLATMC8416 (PLA/TMC ratio: 84:16; M(w): 170 kDa) films. Furthermore, when the BFB0261-loaded films constructed from various polymers were implanted subcutaneously on rat backs, the PLAPEG8515H and PLATMC8416 films were capable of achieving sustained release of BFB0261 at the administrated site for 12 weeks. Therefore, the present data indicate that films constructed from PLAPEG8515H or PLATMC8416 may be applicable to bone or tissue engineering.     

Copolymers of poly(lactic acid) and d-α-tocopheryl polyethylene glycol 1000 succinate-based nanomedicines: versatile multifunctional platforms for cancer diagnosis and therapy

Introduction: The major drawbacks associated with most of the anti-cancer drugs are their potential adverse effects. Distribution of these drugs throughout the body causes untoward adverse effects and less accumulation of drug at the site of tumors also causes decrease in therapeutic efficacy. Targeted nanomedicines are the emerging systems to improve the targetability of drug to the tumor site and to reduce the toxicity with maximum efficacy. Copolymers of poly-lactic acid (PLA) and d-α-tocopheryl polyethylene glycol 1000 succinate (Vitamin-E TPGS or TPGS) are innovative materials being actively investigated for the fabrication of non-targeted and targeted nanomedicines for diagnosis and therapy of cancer.

Areas covered: In this review, different nanomedicines of copolymers such as poly-lactic acid – polyoxyethylene sorbitan monooleate (PLA – Tween® 80), poly-lactic acid – poly-ethyleneglycol (PLA-PEG), poly-lactic acid-d-α-tocopheryl polyethylene glycol 1000 succinate (PLA-TPGS) and TPGS-based nanomedicines (i.e., TPGS emulsified polymeric nanoparticles, TPGS prodrugs, TPGS liposomes, and TPGS micelles) for the diagnosis and therapy of cancer have been discussed.

Expert opinion: PLA, PLA-Tween® 80, PLA-PEG, PLA-TPGS, and TPGS are the promising polymeric biomaterials well studied as cancer nanomedicines. These biomaterials have proved that they could be applied in the fabrication of multifunctional nanomedicines for the future needs in simultaneous diagnosis of cancer as well as targeted chemotherapy.     

Optimization of PEGylated nanoemulsions for improved pharmacokinetics of BCS class II compounds

Abstract

The objective of the study was the optimization of nanoemulsion formulations to prevent their rapid systemic clearance after intravenous administration. An amphiphilic PEG derivative DSPE-PEG (1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-methoxy-poly(polyethylene glycol) with different chain lengths and concentration was used as a nanoemulsion droplet surface modifier. The danazol loading in all nanoemulsions was kept on the same level of ～2?mg/mL. In the present investigation, PEGylated and non-PEGylated nanoemulsions were compared in vitro phagocytosis by incubating with lung macrophages and in vivo after intravenous administration in rats. Danazol-containing nanoemulsions (NE) modified with various PEG chain lengths (2000–10?000) and concentrations (3–12?mg/mL) were prepared and characterized. Nanoemulsion droplets were reproducibly obtained in the size range of 213–340?nm. The non-PEGylated NE had the surface charge of ?25.4?mV. This absolute charge value decreased with increasing chain length and concentration. With increase in chain length and density the macrophage uptake decreased which could be due to decrease in surface charge and hydrophilicity of droplets. The greatest shielding of the NE droplets was reached with DSPE-PEG₅₀₀₀ at the concentration of 6?mg/mL where the surface charge changed to ?1.27?mV. Following intravenous administration a maximum danazol exposure (401?±?68.2?h?ng/mL) was observed with the lowest clearance rate (5.06?±?0.95?L/h/kg) from 6?mg/mL DSPE-PEG₅₀₀₀ nanoemulsion. PEG₅₀₀₀ and PEG₁₀₀₀₀ altered the pharmacokinetic of danazol by decreasing clearance and volume of distribution which is likely explained by the presence of hydrophilic shields around the droplets that prevent their rapid systemic clearance and tissue partitioning.     

Modification of the Copolymers Poloxamer 407 and Poloxamine 908 can Affect the Physical and Biological Properties of Surface Modified Nanospheres

Purpose. To investigate the effects of the modification of the copolymers poloxamer 407 and poloxamine 908 on the physical and biological properties surface modified polystyrene nanospheres. Methods. A method to modify poloxamer 407 and poloxamine 908, introducing a terminal amine group to each PEO chain has been developed. The aminated copolymers can be subsequently radiolabelled with lodinated (I¹²⁵) Bolton-Hunter reagent. The aminated copolymers were used to surface modify polystyrene nanospheres. The physical and biological properties of the coated nanospheres were studied using particle size, zeta potential, in vitro non-parenchymal cell uptake and in vivo biodistribution experiments. Results. The presence of protonated amine groups in the modified copolymers significantly affected the physical and biological properties of the resulting nanospheres, although the effects were copolymer specific. The protonated surface amine groups in both copolymers reduced the negative zeta potential of the nanospheres. Acetylation of the copolymer's free amine groups resulted in the production of nanospheres with comparable physical properties to control unmodified copolymer coated nanospheres. In vivo, the protonated amine groups in the copolymers increased the removal of the nanospheres by the liver and spleen, although these effects were more pronounced with the modified poloxamer 407 coated nanospheres. Acetylation of the amine groups improved the blood circulation time of the nanospheres providing modified poloxamine 908 coated nanospheres with comparable biological properties to control poloxamine 908 coated nanospheres. Similarly, modified poloxamer 407 coated nanospheres had only slightly reduced circulation times in comparison to control nanospheres. Conclusions. The experiments have demonstrated the importance of copolymer structure on the biological properties of surface modified nanospheres. Modified copolymers, which possess comparable properties to their unmodified forms, could be used in nanosphere systems where antibody fragments can be attached to the copolymers, thereby producing nanospheres which target to specific body sites.     

Graphene oxide stabilized by PLA–PEG copolymers for the controlled delivery of paclitaxel

PurposeTo investigate the application of water-dispersible poly(lactide)–poly(ethylene glycol) (PLA–PEG) copolymers for the stabilization of graphene oxide (GO) aqueous dispersions and the feasibility of using the PLA–PEG stabilized GO as a delivery system for the potent anticancer agent paclitaxel.MethodsA modified Staudenmaier method was applied to synthesize graphene oxide (GO). Diblock PLA–PEG copolymers were synthesized by ring-opening polymerization of dl-lactide in the presence of monomethoxy-poly(ethylene glycol) (mPEG). Probe sonication in the presence of PLA–PEG copolymers was applied in order to reduce the hydrodynamic diameter of GO to the nano-size range according to dynamic light scattering (DLS) and obtain nano-graphene oxide (NGO) composites with PLA–PEG. The composites were characterized by atomic force microscopy (AFM), thermogravimetric analysis (TGA), and DLS. The colloidal stability of the composites was evaluated by recording the size of the composite particles with time and the resistance of composites to aggregation induced by increasing concentrations of NaCl. The composites were loaded with paclitaxel and the in vitro release profile was determined. The cytotoxicity of composites against A549 human lung cancer cells in culture was evaluated by flow cytometry. The uptake of FITC-labeled NGO/PLA–PEG by A549 cells was also estimated with flow cytometry and visualized with fluorescence microscopy.ResultsThe average hydrodynamic diameter of NGO/PLA–PEG according to DLS ranged between 455 and 534 nm, depending on the molecular weight and proportion of PLA–PEG in the composites. NGO/PLA–PEG exhibited high colloidal stability on storage and in the presence of high concentrations of NaCl (far exceeding physiological concentrations). Paclitaxel was effectively loaded in the composites and released by a highly sustained fashion. Drug release could be regulated by the molecular weight of the PLA–PEG copolymer and its proportion in the composite. The paclitaxel-loaded composites exhibited cytotoxicity against A549 cancer cells which increased with incubation time, in conjunction with the increasing with time uptake of composites by the cancer cells.ConclusionGraphene oxide aqueous dispersions were effectively stabilized by water-dispersible, biocompatible and biodegradable PLA–PEG copolymers. The graphene oxide/PLA–PEG composites exhibited satisfactory paclitaxel loading capacity and sustained in vitro drug release. The paclitaxel-loaded composites could enter the A549 cancer cells and exert cytotoxicity. The results justify further investigation of the suitability of PLA–PEG stabilized graphene oxide for the controlled delivery of paclitaxel.     

Stabilization and Controlled Release of Bovine Serum Albumin Encapsulated in Poly(D,L-lactide) and Poly(ethylene glycol) Microsphere Blends

Purpose. The acidic microclimate in poly(D, L-lactide-co-glycolide) 50/50 microspheres has been previously demonstrated by our group as the primary instability source of encapsulated bovine serum albumin (BSA). The objectives of this study were to stabilize the encapsulated model protein, BSA, and to achieve continuous protein release by using a blend of: slowly degrading poly(D, L-lactide) (PLA), to reduce the production of acidic species during BSA release; and pore-forming poly(ethylene glycol) (PEG), to increase diffusion of BSA and polymer degradation products out of the polymer. Methods. Microspheres were formulated from blends of PLA (Mw 145,000) and PEG (Mw 10,000 or 35,000) by using an anhydrous oil-in-oil emulsion and solvent extraction (O/O) method. The polymer blend composition and phase miscibility were examined by FT-IR and DSC, respectively. Microsphere surface morphology, water uptake, and BSA release kinetics were also investigated. The stability of BSA encapsulated in microspheres was examined by losses in protein solubility, SDS-PAGE, IEF, CD, and fluorescence spectroscopy. Results. PEG was successfully incorporated in PLA microspheres and shown to possess partial miscibility with PLA. A protein loading level of 5% (w/w) was attained in PLA/PEG microspheres with a mean diameter of approximately 100 m. When PEG content was less than 20% in the blend, incomplete release of BSA was observed with the formation of insoluble, and primarily non-covalent aggregates. When 20%-30% PEG was incorporated in the blend formulation, in vitro continuous protein release over 29 days was exhibited. Unreleased BSA in these formulations was water-soluble and structurally intact. Conclusions. Stabilization and controlled relaease of BSA from PLA/PEG microspheres was achieved due to low acid and high water content in the blend formulation.     

Development of Systems for Targeting the Regional Lymph Nodes for Diagnostic Imaging: In Vivo Behaviour of Colloidal PEG-Coated Magnetite Nanospheres in the Rat Following Interstitial Administration

Purpose. Nanoparticles can be utilised for targeting drugs to the regional lymph nodes or as diagnostic agents. The surface modification of magnetite nanospheres with poly(ethylene glycol) (PEG) has been assessed by in vitro characterisation and in vivo studies following subcutaneous administration to the rat. Methods. Magnetite nanospheres were prepared with a grafted PEG layer using various PEG lengths from 350 to 1000 Da. Thermogravimetric analysis was utilised to measure the adsorbed amount of PEG. Colloid stability was confirmed by measurement of the particle size and electrophoretic mobility. The kinetics of injection site drainage and lymph node retention were determined 2 hours after subcutaneous administration, for nanospheres coated with PEG lengths of 350, 550, 750, and 1000 Da. For the 750 PEG coated nanospheres, the kinetics of distribution was determined over a 48–hour time course. Results. The distribution of the nanospheres was modified and the lymph node localisation enhanced by altering the surface coverage of PEG on the magnetic surface. Conclusions. PEG–coated magnetite nanospheres with different surface characteristics can be utilised to target a diagnostic agent to regional lymph nodes.     

Self-assembled amphiphilic hyaluronic acid graft copolymers for targeted release of antitumoral drug

Polymeric micelles obtained by self-assembling of amphiphilic hyaluronic acid (HA) graft copolymers have been prepared and characterized. In particular, hyaluronic acid (HA) has been grafted to polylactic acid (PLA) and polyethylenglycol chains (PEG), then the copolymers able to form micelles in aqueous medium have been chosen to entrap the antitumoral drug Doxorubicin. The critical aggregation concentration of HA-g-PLA or HA-g-PLA-g-PEG micelles has been determined by using pyrene as a fluorescent probe, whereas their shape and size have been evaluated by light scattering measurements, scanning and transmission electron microscopies. The selective cytotoxicity of drug loaded micelles toward the CD-44 over-expressing HCT-116 cells compared to receptor deficient human derm fibroblasts has been demonstrated. Pegylated micelles showed better stability and drug loading capacity and they were able to escape from macrophage phagocytosis.     

Preparation and in vivo evaluation of anti-plasmodial properties of artemisinin-loaded PCL–PEG–PCL nanoparticles

Abstract

Purpose: Artemisinin (ART) has anti-inflammatory, antimicrobial, antioxidant, anti-amyloid, and anti-malarial effects, but its application is limited due to its low water solubility and poor oral bioavailability. In this study, the bioavailability, water solubility, and anti-plasmodial property of ART were improved by PCL–PEG–PCL tri-block copolymers.

Methods: The structure of the copolymers was characterized by ¹H NMR, FT-IR, DSC, and GPC techniques. ART was encapsulated within micelles by a single-step nano-precipitation method, leading to the formation of ART-loaded PCL–PEG–PCL micelles. The obtained micelles were characterized by dynamic light scattering (DLS) and atomic force microscopy (AFM). The in vivo anti-plasmodial activity of ART-loaded micelles was measured against Plasmodium berghei infected Swiss albino mice.

Results: The results showed that the zeta potential of ART-loaded micelles was about ?8.37?mV and the average size was 91.87?nm. ART was encapsulated into PCL–PEG–PCL micelles with a loading capacity of 19.33?±?0.015% and encapsulation efficacy of 87.21?±?3.32%. In vivo anti-plasmodial results against P. berghei showed that multiple injections of ART-loaded micelles could prolong the circulation time and increase the therapeutic efficacy of ART.

Conclusion: These results suggested that PCL–PEG–PCL micelles would be a potential carrier for ART for the treatment of malaria.     

Self-assembled filomicelles prepared from polylactide-poly(ethylene glycol) diblock copolymers for sustained delivery of cycloprotoberberine derivatives

Polylactide-poly(ethylene glycol) (PLA-PEG) block copolymers were synthesized by ring opening polymerization of l-lactide using a monomethoxy PEG (mPEG) as macroinitiator and zinc lactate as catalyst. The resulting diblock copolymers were characterized by ¹H NMR and GPC. Polymeric micelles were prepared by self-assembly of copolymers in distilled water using co-solvent evaporation or membrane hydration methods. The resulting micelles are worm-like in shape as shown by TEM measurements. A hydrophobic anticancer drug, cycloprotoberberine derivative A35, was successfully loaded in PLA-PEG filomicelles with high encapsulation efficiency (above 88%). Berberine (BBR) was studied for comparison. In both methods, PLA-PEG filomicelles were prepared with a theoretical loading of 5%, 10% and 20%. Physical stability studies indicated that BBR/A35-loaded filomicelles were more stable when stored at 4?°C than at 25?°C. Compared with BBR-loaded filomicelles, A35-loaded filomicelles exhibited higher antitumor activity. Importantly, the in vitro cytotoxicity and stability of A35-loaded filomicelles evidenced the potential of drug-loaded filomicelles in the development of drug delivery systems.     

VEGF-controlled release within a bone defect from alginate/chitosan/PLA-H scaffolds

VEGF and its receptors constitute the key signaling system for angiogenic activity in tissue formation, but a direct implication of the growth factor in the recruitment, survival and activity of bone forming cells has also emerged. For this reason, we developed a composite (alginate/chitosan/PLA-H) system that controls the release kinetics of incorporated VEGF to enhance neovascularization in bone healing. VEGF release kinetics and tissue distribution were determined using iodinated (¹²⁵I) growth factor. VEGF was firstly encapsulated in alginate microspheres. To reduce the high in vitro burst release, the microspheres were included in scaffolds. Matrices were prepared with alginate (A-1, A-2), chitosan (CH-1, CH-2) or by coating the CH-1 matrix with a PLA-H (30 kDa) film (CH-1-PLA), the latter one optimally reducing the in vitro and in vivo burst effect. The VEGF in vitro release profile from CH-1-PLA was characterized by a 13% release within the first 24 h followed by a constant release rate throughout 5 weeks. For VEGF released from composite scaffolds in vitro, bioactivity was maintained above 90% of the expected value. Despite the fact that the in vivo release rate was slightly faster, a good in vitro–in vivo correlation was found. The VEGF released from CH-1 and CH-1-PLA matrices implanted into the femurs of rats remained located around the implantation site with a negligible systemic exposure. These scaffolds provided a bone local GF concentration above 10 ng/g during 2 and 5 weeks, respectively, in accordance to the in vivo release kinetics. Our data show that the incorporation of VEGF into the present scaffolds allows for a controlled release rate and localization of the GF within the bone defect.     

Noninvasive Visualization of Pharmacokinetics,Biodistribution and Tumor Targeting of Poly[<Emphasis Type="BoldItalic">N</Emphasis>-(2-hydroxypropyl)methacrylamide] in Mice Using Contrast Enhanced MRI

Purpose To study a non-invasive method of using contrast enhanced magnetic resonance imaging (MRI) to visualize the real-time pharmacokinetics, biodistribution and tumor accumulation of paramagnetically labeled poly[N-(2-hydroxypropyl)methacrylamide] (PHPMA) copolymer conjugates with different molecular weights and spacers in tumor-bearing mice. Materials and Methods Paramagnetically labeled HPMA copolymer conjugates were synthesized by free radical copolymerization of HPMA with monomers containing a chelating ligand, followed by complexation with Gd(OAc)₃. A stable paramagnetic chelate, Gd-DO3A, was conjugated to the copolymers via a degradable spacer GlyPheLeuGly and a non-degradable spacer GlyGly, respectively. The conjugates with molecular weights of 28, 60 and 121 kDa and narrow molecular weight distributions were prepared by fractionation with size exclusion chromatography. The conjugates were injected into athymic nude mice bearing MDA-MB-231 human breast carcinoma xenografts via a tail vein. MR images were acquired before and at various time points after the injection with a 3D FLASH sequence and a 2D spin-echo sequence at 3T. Pharmacokinetics, biodistribution and tumor accumulation of the conjugates were visualized based on the contrast enhancement in the blood, major organs and tumor tissue at various time points. The size effect of the conjugates was analyzed among the conjugates. Results Contrast enhanced MRI resulted in a real-time, three-dimensional visualization of blood circulation, pharmacokinetics, biodistribution and tumor accumulation of the conjugates, and the size effect on these pharmaceutical properties. HPMA copolymer conjugates with high molecular weight had a prolonged blood circulation time and high passive tumor targeting efficiency. Non-biodegradable HPMA copolymers with molecular weights higher than the threshold of renal filtration demonstrated higher efficiency for tumor drug delivery than biodegradable poly(L-glutamic acid). Conclusions Contrast enhanced MRI is an effective method for non-invasive visualization of in vivo properties of the paramagnetically labeled polymer conjugates in preclinical studies.