Biodegradable Delivery System Research Articles

Nanoparticle formulation enhances the delivery and activity of a vascular endothelial growth factor antisense oligonucleotide in human retinal pigment epithelial cells.

The objective of this study was to investigate the delivery and activity of a vascular endothelial growth factor (VEGF) antisense oligonucleotide in a human retinal pigment epithelial cell line (ARPE-19) using a biodegradable nanoparticulate delivery system. A 19-mer antisense phosphorothioate oligonucleotide (PS-ODN) complementary to bases 6-24 relative to the translational start site of the VEGF mRNA, a sense PS-ODN and a mismatch PS-ODN were examined for the inhibition of secretion and mRNA expression of VEGF using an enzyme-linked immunosorbent assay and reverse transcription-polymerase chain reaction, respectively. Nanoparticles of the antisense oligonucleotides were formulated using a poly(lactide-co-glycolide) (50:50) copolymer using a double emulsion solvent evaporation method. After preparing nanoparticles, drug loading, encapsulation efficiency and particle size were determined. The cells were exposed to either plain solution of oligonucleotide or nanoparticles of oligonucleotide from Day 3 through Day 6. Alternatively, the cells were incubated with PS-ODNs and lipofectin for 4 h on Day 4. In all studies, VEGF secretion and mRNA expression were determined on Day 6. The particle size, drug loading and encapsulation efficiency were 252 nm, 5.5% and 16.5%, respectively. The antisense PS-ODN inhibited VEGF mRNA and protein secretion when delivered using nanoparticles or lipofectin but not in its free form. This was consistent with the ability of nanoparticles and lipofectin to elevate the cellular uptake of the oligonucleotide by 4-fold and 13-fold, respectively. Neither mismatch nor sense oligonucleotides inhibited VEGF secretion. In conclusion, biodegradable nanoparticles enhance cellular delivery of a VEGF antisense oligonucleotide and inhibit VEGF secretion and mRNA expression in a human retinal pigment epithelial cell line.

Encapsulation into amphiphilic polyanhydride microparticles stabilizes Yersinia pestis antigens

The design of biodegradable polymeric delivery systems based on polyanhydrides that would provide for improved structural integrity of Yersinia pestis antigens was the main goal of this study. Accordingly, the full-length Y. pestis fusion protein (F1–V) or a recombinant Y. pestis fusion protein (F1B2T1–V10) was encapsulated and released from microparticles based on 1,6-bis(p-carboxyphenoxy)hexane (CPH) and sebacic acid (SA) copolymers and 1,8-bis(p-carboxyphenoxy)-3,6-dioxaoctane (CPTEG) and CPH copolymers fabricated by cryogenic atomization. An enzyme-linked immunosorbent assay was used to measure changes in the antigenicity of the released proteins. The recombinant F1B2T1–V10 was unstable upon release from the hydrophobic CPH:SA microparticles, but maintained its structure and antigenicity in the amphiphilic CPTEG:CPH system. The full-length F1–V was stably released by both CPH:SA and CPTEG:CPH microparticles. In order to determine the effect of the anhydride monomers on the protein structure, changes in the primary, secondary, and tertiary structure, as well as the antigenicity of both Y. pestis antigens, were measured after incubation in the presence of saturated solutions of SA, CPH, and CPTEG anhydride monomers. The results indicated that the amphiphilic environment provided by the CPTEG monomer was important to preserve the structure and antigenicity of both proteins. These studies offer an approach by which a thorough understanding of the mechanisms governing antigenic instability can be elucidated in order to optimize the in vivo performance of biodegradable delivery devices as protein carriers and/or vaccine adjuvants.

Novel reducible linear L-lysine-modified copolymers as efficient nonviral vectors

The development of biodegradable gene delivery systems with high transfection efficiencies is paramount to the clinical translation of nonviral gene carriers. Therefore, to produce a biocompatible, reducible, effective and non-toxic gene delivery system, we have designed and synthesized novel reducible linear L-lysine modified copolymers (LLCs) as an alternative to high molecular weight poly(L-lysine) (PLL). The molecular weight (MW) of the copolymers was found to be ∼ 3.2 kDa with a polydispersity index of ∼ 1.2. Gel retardation assays showed complete condensation of DNA at N/P ratios greater than 20/1 and exceptional LLC/pDNA polyplex stability during incubation with DNase I. Release of DNA from the polyplexes only occurred in the presence of the reducing agent dithiothreitol (DTT). The particle sizes of LLC/pDNA polyplexes were found to be between 100 and 231 nm with surface charges of 0.8–17 mV respectively. The transfection efficiencies of the polyplexes as determined with a luciferase assay showed that LLC polyplexes produced five times higher transfection efficiencies in HDF cells, three times higher transfection efficiencies in MCF-7 cells, and four times higher transfection efficiencies in MA cells as compared to the optimal PLL control. The LLC/pDNA polyplexes showed significantly lower cytotoxicities as compared to the control in HDF, MCF-7 and MA cells at certain N/P ratios. Therefore, these results suggest that these novel LLCs are efficient, reducible and biocompatible polymers for nonviral gene delivery.

A biodegradable antibiotic delivery system based on poly-(trimethylene carbonate) for the treatment of osteomyelitis

Background and purpose Many investigations on biodegradable materials acting as an antibiotic carrier for local drug delivery are based on poly(lactide). However, the use of poly(lactide) implants in bone has been disputed because of poor bone regeneration at the site of implantation. Poly(trimethylene carbonate) (PTMC) is an enzymatically degradable polymer that does not produce acidic degradation products. We explored the suitability of PTMC as an antibiotic releasing polymer for the local treatment of osteomyelitis.Methods This study addressed 2 separate attributes of PTMC: (1) the release kinetics of gentamicin-loaded PTMC and (2) its behavior in inhibiting biofilm formation. Both of these characteristics were compared with those of commercially available gentamicin-loaded poly(methylmethacrylate) (PMMA) beads, which are commonly used in the local treatment of osteomyelitis.Results In a lipase solution that mimics the in vivo situation, PTMC discs with gentamicin incorporated were degraded by surface erosion and released 60% of the gentamicin within 14 days. This is similar to the gentamicin release from clinically used PMMA beads. Moreover, biofilm formation by Staphylococcus aureus was inhibited by approximately 80% over at least 14 days in the presence of gentamicin-loaded PTMC discs. This is similar to the effect of gentamicin-loaded PMMA beads. In the absence of the lipase, surface erosion of PTMC discs did not occur and gentamicin release and biofilm inhibition were limited.Interpretation Since gentamicin-loaded PTMC discs show antibiotic release characteristics and biofilm inhibition characteristics similar to those of gentamicin-loaded PMMA beads, PTMC appears to be a promising biodegradable carrier in the local treatment of osteomyelitis.

Amine-modified hyperbranched polyesters as non-toxic, biodegradable gene delivery systems

For chronic non-viral gene therapy, biodegradable carriers with low cytotoxicity are essential. To create a series of non-toxic and biodegradable gene carriers, hyperbranched polymers based on 2,2-bis-(methylol)propionic acid (bis-MPA); (Boltorn H ®) were modified by introducing tertiary amines. The terminal OH groups were modified with diethylaminopropylamine (DEAPA) by carbonyldiimidazole (CDI) chemistry. The resulting polymers were characterized by 1H, 13C NMR, IR and GPC. Degradability and degradation rate were investigated with respect to the degree of amine substitution. The toxicity of all hyperbranched polyesters was generally very low compared to polyethyleneimine (PEI). Measurements of size and zeta potential showed that small nano-complexes with a positive zeta potential were formed. Dependency of the degree of amine substitution on interaction with DNA was studied by agarose gel retardation assay and ethidium bromide exclusion assay. Influence of the amine substitution on transfection efficiency of the different polymers demonstrated that a certain amine substitution degree was required to achieve transfection efficiency. These carriers provide degradability, very low toxicity and the ability to transfect cells which can be influenced by the degree of amine substitution.

A surface-eroding antibiotic delivery system based on poly-(trimethylene carbonate)

Biodegradable delivery systems that do not produce acidic compounds during degradation are preferred for local antibiotic delivery in bone infections in order to avoid adverse bone reactions. Poly(trimethylene carbonate) (PTMC) has good biocompatibility, and is such a polymer. The objective of this in vitro study was to explore the suitability of PTMC as an antibiotic releasing polymer for the local treatment of bone infections. Degradation behaviour and corresponding release profiles of gentamicin and vancomycin from slowly degrading PTMC168 and faster degrading PTMC339 discs were compared in the absence and presence of a lipase solution. Gentamicin release in the absence of lipase was diffusion-controlled, while vancomycin release was limited. Surface erosion of PTMC only occurred in the presence of lipase. Both antibiotics were released in high concentrations from PTMC in the presence of lipase through a combination of surface erosion and diffusion. This illustrates the major advantage of surface-eroding biodegradable polymers, allowing release of larger antibiotic molecules like vancomycin.

Intra-operative cisplatin for the treatment of canine extremity soft tissue sarcomas

Nineteen dogs with histologically confirmed soft tissue sarcomas of the extremities were treated with a combination of marginal surgery and intra-operative chemotherapy in the form of cisplatin in a biodegradable implant delivery system (Atrigel; Atrix Laboratories, Fort Collins, Co, USA). None of the dogs had evidence of metastasis at time of treatment. The median dose of cisplatin was 52.1 mg/m(2) (mean 55.4 mg/m(2), range 18.5-108.6 mg/m(2)). Wound complications were noted in 16 dogs (84.2%). Median follow-up time was 874 days (mean 777 days, range 125-1463 days). Nine dogs (47.3%) were alive at the time of analysis. Local recurrence occurred in three dogs (16.6%). The time to recurrence was 214, 264 and 874 days.

Fabrication of cefuroxime-impregnated calcium sulfate: Polycaprolactone composite implant for osteomyelitis

Osteomyelitis is characterized as an inflammatory bone disease caused by pyrogenic bacteria. As oral bioavailabilities of antibiotics are low, a regimen of 6 weeks of intravenous antibiotic is necessary for adequate therapy. Although the dose of antibiotic administered systemically is high, therapeutically effective drug concentrations are not always achieved at the site of infection. This problem can be overcome by the use of local antibiotics from a biodegradable implant for chronic osteomyelitis that can deliver the drug at least for 6 weeks.The implant delivers high antibiotic concentration at tissue levels, obliterates dead space, aids bone repair and does not need to be removed. The aim of this study was to develop and evaluate a calcium sulfate and polycaprolactone (PCL)-based composite biodegradable implantable delivery system of cefuroxime for the localized treatment of osteomyelitis that can deliver the drug for at least 6 weeks.The PCL and calcium sulfate composite system has not been studied yet. Interaction studies were carried out to check any incompatibility between the ingredients. Implants were prepared by a modified fabrication technique to avoid solvent use. The prepared implants were evaluated for various in vitro parameters like dimensions, hardness, tensile strength, drug release profile, sterility test and morphological changes in pellet before and after drug release. The pellets were also tested for microbiological efficacy and compared with a plain drug solution in different concentrations. Developed pellets are regular in shape and size with good tensile strength. The release profile displayed drug levels above the minimum inhibitory concentration continuously for up to 2 months. A wide zone of inhibition by the pellet against Staphylococcus aureus as compared with the drug solution proves its efficacy in the treatment of osteomyelitis. Results show that the developed calcium sulfate and PCL-based composite biodegradable implantable delivery system of cefuroxime is a good alternate system and can deliver the drug for more than 6 weeks, maintaining an adequate inhibitory concentration at the site.

Cefoperazone sodium impregnated polycaprolactone composite implant for osteomyelitis

The use of local antibiotics from a biodegradable implant for chronic osteomyelitis is an attractive alternative. The implant delivers high antibiotic concentration at tissue levels, obliterates dead space, aids bone repair and does not need to be removed. The purpose of this paper is to develop and evaluate a calcium sulphate and polycaprolactone based composite biodegradable implantable delivery system of cefoperazone sodium. Implants were prepared by modified fabrication technique to avoid solvent use. Interaction studies were carried out to check any incompatibility between ingredients. Prepared implants were evaluated for various in vitro parameters like dimensions, hardness, tensile strength, drug release profile and sterility. Morphological changes in pellet before and after drug release were evaluated by scanning electron microscopy. The pellet were also tested for microbiological efficacy and compared with plain drug solution in different concentrations. Developed pellets are regular in shape and size with good tensile strength. The release profile displayed drug levels above MIC continuously up to 2 months. Wide zone of inhibition by pellet against Staph. aureus as compared to drug solution proves its efficacy in treatment of osteomyelitis.

Investigation of Drug Release from Biodegradable Polymeric Delivery System by Infrared Spectrometry

This study investigates some effective parameters in betamethasone release from in situ forming biodegradable drug delivery systems. The drug delivery systems are based on two biodegradable polymers of poly(D,L-lactide-co-glycolide) (PLGA), which are commercially called RG504h and RG756. Temperature, polymer type, and γ-irradiation are the parameters investigated by attenuated total reflectance Fourier transform-infrared (ATR-FTIR) spectrometry in the 1891–1324 cm−1 spectral region. Obtained results were compared with those of the HPLC method, demonstrating several similarities. N-methyl-2-pyrrolidinone (NMP) was employed as solvent. The γ-radiation (25 kGy) has no effect on decomposition of betamethasone, while it increases the rate of drug release. RG756 reduces the release rate in comparison with RG504H. Temperature does not affect the amount of free drug in the polymer matrix. The partial least squares (PLS) regression method with path length constant, mean centering, and variance scaling techniques was applied. Correlation coefficient R2 and root mean square error of calibration (RMSEC) were 0.998 and 0.106 respectively.

Biodegradable gentamicin delivery systems for parenteral use for the treatment of intracellular bacterial infections

Gentamicin is an aminoglycoside with a wide spectrum of antibacterial activity. However, as a highly water-soluble drug, it penetrates cells poorly. This constitutes a particularly important drawback for treating intracellular bacterial infections. This major hurdle may be solved by the use of vectors to deliver and target bioactive agents to the intracellular sites of infection. Thus, in the case of antimicrobials, drug delivery systems may help to increase their therapeutic index in intracellular locations. The development and evolution of pharmaceutical forms of gentamicin for the parenteral treatment of intracellular pathogens is reviewed in this paper.

Controlled release of vancomycin from cross-linked gelatine

This paper explores the possibility of using biodegradable cross-linked gelatines as antibiotic devices for a long-term elution (80 days). Capillary electrophoresis (CE) has been utilized to evaluate the mass percentage of vancomycin and gelatine contemporary released from differently cross-linked vancomycin loaded gelatine samples in an elution time ranging from 24 to 1920 h. While the solubilization kinetic of gelatine samples differently cross-linked can be very close described by the simplified Higuchi model, the vancomycin release kinetic is contemporary governed by both the Fickian diffusion process trough the gelatine matrix network and the dissolution process of the matrix due to its degradation. Comparing the antibiotic eluting kinetics from gelatine at diverse cross-linking degree we observed that the degradation of the proteic matrix appears to have a minor influence in the drug release control. Vancomycin released from all the gelatine partially cross-linked samples results active against Staphylococcus aureus and Streptococcus faecalis which represent the most pathogens commonly isolated in orthopaedic infections. Vancomycin overcomes the minimum inhibitory concentration for both the bacteria in the whole range of elution time. Cross-linked gelatine devices appear to represent a useful biodegradable delivery system for local anti-infective therapy in arthoplasty.

Reconstruction of Bone Defects Using rhBMP-2-coated Devitalized Bone

Massive bone defects often are caused by radical resection of bone tumors. Reconstruction of the defect by reimplantation of the resected bone segment after it has been devitalized is advantageous because of its ability to match the size of the defect. In addition, this technique carries a low risk for local recurrence of the tumor, avoids immunologic reaction, and is low in cost. However, limited osteogenic potential of the devitalized bone often leads to delayed union, gradual resorption, and mechanical weakness of the reimplanted segment. We applied rhBMP-2 in a biodegradable polymer delivery system to the devitalized bone. Middiaphyseal bone defects were created by resection in rat femurs. The resected segments were autoclaved at 135 degrees C for 15 minutes, coated with a rhBMP-2-retaining paste on the outer surface, and then reimplanted into the defects. In a brief time, newly formed bone was seen on the surface of the devitalized bone. After 12 weeks, a solid bone mass encasing the dead bone segments was consistently formed and abundant new bone formation was visible in the segments as they were remodeled. The amount of new bone formed could be regulated by the amount of the rhBMP-2-retaining paste applied to the bone segments. This method presents a new approach for the reconstruction of bone defects.

The effect of paclitaxel-loaded nanoparticles with radiation on hypoxic MCF-7 cells

The inability of radiotherapy to eradicate completely certain human tumours may be due to the presence of resistant hypoxic cells. Several studies have confirmed the radiosensitizing effect of paclitaxel, a microtubular inhibitor. The object of this study was to evaluate the physicochemical characteristics of paclitaxel-loaded nanoparticles, and determine the ability of the released paclitaxel to radiosensitize hypoxic human breast carcinoma cells (MCF-7) with respect to radiation dose. The poly(d,l-lactide-co-glycolide) (PLGA) nanoparticles containing paclitaxel were prepared by o/w emulsification-solvent evaporation method. The morphology of the paclitaxel-loaded nanoparticles was investigated by scanning electron microscopy. The drug encapsulation efficiency (EE) and in vitro release profile were measured by high-performance liquid chromatography. Cell cycle was evaluated by flow cytometry. Cell viability was measured by the ability of single cells to form colonies in vitro. The prepared nanoparticles were spherical with diameter between 200 and 800 nm. The EE was 85.5%. The drug release pattern was biphasic with a fast release rate followed by a slow one. Co-culture of human breast carcinoma cells (MCF-7) with paclitaxel-loaded nanoparticles demonstrated that released paclitaxel retained its bioactivity to block cells in the G2/M phase of the cell cycle and effectively sensitized hypoxic MCF-7 cells to radiation with radiosensitivity shown to be dependent of radiation dose at levels of dosages studied. The sensitizer enhancement ratio for paclitaxe-loaded nanoparticles at 10% survival is approximately 1.4. This work has demonstrated that paclitaxel can be effectively released from a biodegradable PLGA nanoparticle delivery system while maintaining potent combined cytotoxic and radiosensitizing abilities for hypoxic tumour cells.

Gatifloxacin Biodegradable Implant for Treatment of Experimental Osteomyelitis: In Vitro and In Vivo Evaluation

Osteomyelitis is an inflammatory bone disease caused by pyogenic bacteria. The advantages of localized biodegradable therapy for osteomyelitis include high local antibiotic concentration at the site of infection and obviation of the need for removal of the implant after treatment. The purpose of this study was to develop and evaluate a biodegradable implantable delivery system containing gatifloxacin (GAT) for the localized treatment of osteomyelitis, experimentally induced by methicillin resistant Staphylococcus aureus (MRSA). Implants, prepared by solvent casting technique, showed reasonable tensile strength. DSC examination indicated that GAT is present in an amorphous form in the implant. The in vitro release of GAT showed a profile characterized by an initial burst followed by a second stage of gradual delivery over 27 days. The in vivo release study revealed that GAT concentrations achieved during the first 3 weeks after implantation exceeded the MIC of GAT against MRSA by > 100,000 times. Bacterial tibial bone count performed in rabbits tibia 2 and 4 weeks after implantation of GAT implant in infected bone indicated complete eradication of infection in all treated rabbits as indicated by the significant decrease in bacterial count. The results show that the proposed implant may have a promising role in the therapeutic approach to osteomyelitis.

Application of control-release preparations of growth factor in treating neurodegeneration diseases

利用缓释系统包载生长因子,既能保护生长因子的生物活性,又可以使其缓慢释放.从载体材料与生长因子的简单混合到生长因子缓释微球系统,生长因子缓释技术不断更新并得到广泛应用.就生物可降解生长因子缓释制剂在神经系统退变性疾病中的应用做一综述。

Development of a biodegradable alginate carrier system for antibiotics and bone cells

This study presents a novel biodegradable alginate delivery system for antibiotics and bone cells to treat infected bone defects. About 2 x 10(7) New Zealand rabbit mesenchymal stem cells (MSCs) and 5 mL vancomycin solution (50 mg/mL) were added to 5 mL of 2.5% (w/v) sodium alginate solution to form biodegradable antibiotic and MSCs alginate beads 3 mm in diameter. The alginate beads were then cultured in an osteogenic medium for 14 days. The profiles of antibiotics released from the alginate beads were evaluated using the method of high performance liquid chromatography (HPLC). The expression of osteogenic genes, including Cbfa1 and osteopontin, in the alginate beads was determined by reverse transcription-polymerase chain reaction (RT-PCR) analysis. The alkaline phosphatase activity, calcium level, and mineral deposition of the cultured cells within the alginate beads were assessed. Analytical results demonstrated that the concentrations of vancomycin eluted from the alginate beads were, for 14 days, well above the minimal inhibitory concentration of Staphylococcus aureus. Osteopontin and mRNA of Cbfa1 were detected and increased alkaline phosphatase activity and calcium levels were noted, as was a substantial mineral deposition in cultured MSCs. The PKH 26-labeled MSCs and vancomycin alginate beads were implanted in rabbit bony cavities for in vivo analysis. Implanted PKH 26-labeled MSCs were identified in the newly formed bony trabeculae in all specimens at 2 and 4 months after implantation and there was abundant mineral deposition. The results of in vitro study demonstrated sustained elution of vancomycin from the alginate carrier for 14 days and good osteogenic differentiation of cultured MSCs in the alginate carrier matrix. The results of in vivo study demonstrated the implanted MSCs participating in new bone formation. Based on experimental evidence, development of a biodegradable alginate carrier system for antibiotics and bone cells is possible, providing a potential treatment procedure for infected bone defects.

Local Antibiotic Delivery with OsteoSet??, DBX??, and Collagraft??

Biodegradable local antibiotic delivery systems have gained interest for prophylaxis and treatment of musculoskeletal infections. We studied the biodegradable materials Osteo- Set, DBX and Collagraft for local delivery of vancomycin and gentamicin in vitro. We determined the antimicrobial activity of vancomycin and gentamicin after mixing with each biodegradable material and determined the release of each antimicrobial from each material in an intermittent flow chamber. Antimicrobial activity was expressed as percent of antimicrobial loaded into each sample that was detected; antimicrobial release was expressed as concentration (microg/mL) after timed intervals of chamber flow, peak concentration, area under the curve and percent antimicrobial recovered. Activity of vancomycin after mixing with Osteo- Set, DBX and Collagraft was > 73%. Activity of gentamicin after mixing with DBX was 100%; after mixing with OsteoSet and Collagraft it was reduced to < 61%. AUC0-48hrs of vancomycin was 469, 426 and 432 microg x hr/mL, and the AUC0-48hrs of gentamicin was 368, 306 and 301 microg x hr/mL after release from OsteoSet, DBX, and Collagraft, respectively. Recovered percentages of vancomycin were 39%, 11% and 25%, and recovered percentages of gentamicin were 39%, 9% and 23% after release from OsteoSet, DBX, and Collagraft, respectively. OsteoSet, DBX and Collagraft may be suitable for local delivery of vancomycin and gentamicin.

The role of porous media in biomedical engineering as related to magnetic resonance imaging and drug delivery

Pertinent works associated with magnetic resonance imaging (MRI) and drug delivery are reviewed in this work to demonstrate the role of transport theory in porous media in advancing the progress in biomedical applications. Diffusion process is considered significant in many therapies such as delivering drugs to the brain. Progress in development of the diffusion equation using local volume-averaging technique and evaluation of the applications associated with the diffusion equation are analyzed. Tortuosity and porosity have a significant effect on the diffusion transport. Different relevant models of tortuosity are presented and mathematical modeling of drug release from biodegradable delivery systems are analyzed in this investigation. New models for the kinetics of drug release from porous biodegradable polymeric microspheres under bulk erosion and surface erosion of the polymer matrix are presented in this study. Diffusion of the dissolved drug, dissolution of the drug from the solid phase, and erosion of the polymer matrix are found to play a central role in controlling the overall drug release process. This study paves the road for the researchers in the area of MRI and drug delivery to develop comprehensive models based on porous media theory utilizing fewer assumptions as compared to other approaches.

Influences of excipients on in vitro release and in vivo performance of tetanus toxoid loaded polymer particles

Protein instability during microencapsulation has been one of the major hurdles of biodegradable polymer particles-based vaccine delivery systems. In the present work, effect of serum albumin, sucrose and sodium bicarbonate on surface morphology, entrapment efficiency, in vitro release and in vivo performance tetanus toxoid (TT) loaded PLA particles were investigated. Use of serum albumin as well as high concentration of protein antigen (∼60 mg/ml) helped in protecting the immunoreactivity of the antigen during primary emulsification step of particle formulation. Incorporation of sucrose in the internal aqueous phase led to the reduction in encapsulation efficiency of TT from 43.8 ± 4.3% to 27.3 ± 3.6% in PLA particles and resulted with formation of particles having irregular surface characteristics. Addition of sodium bicarbonate along with sucrose during primary emulsion led to slight improvement in encapsulation efficiency of TT (34.3 ± 3.2%) but affected the in vivo performance in terms of serum anti-TT antibody titers from single point immunization. Restoration of osmotic balance by adding equivalent amount of sucrose in external aqueous phase helped in preventing multiple emulsion instability and subsequently improved the encapsulation efficiency of TT to 63.1 ± 4.2%. Maximum entrapment efficiency of TT up to 69.2 ± 5.1% was achieved when serum albumin, sucrose and sodium bicarbonate were used in internal aqueous phase and sucrose was used in the external aqueous phase. Polymer particles entrapping tetanus toxoid along with optimal stabilizers showed burst release of immunoreactive antigen (>40% in early period) and elicited high and sustained anti-TT antibody titers from single point intramuscular immunization. Anti-TT antibody titers were further enhanced upon immunization of admixture of PLA particles and alum. Choice and use of stabilizers during particle formulation thus need careful considerations not only to protect the immunoreactivity of the antigen, but also to produce stable, uniform particles for optimal in vivo performances.