Design, characterization, and evaluation of intranasal delivery of ropinirole-loaded mucoadhesive nanoparticles for brain targeting.

CONTEXT: Parkinson disease (PD) is a common, progressive neurodegenerative disorder, characterized by marked depletion of striatal dopamine and degeneration of dopaminergic neurons in the substantia nigra. OBJECTIVE: The purpose of the present study was to investigate the possibility of targeting an anti-Parkinson's drug ropinirole (RH) to the brain using polymeric nanoparticles. MATERIALS AND METHODS: Ropinirole hydrochloride (RH)-loaded chitosan nanoparticles (CSNPs) were prepared by an ionic gelation method. The RH-CSNPs were characterized for particle size, polydispersity index (PDI), zeta potential, loading capacity, entrapment efficiency in vitro release study, and in vivo distribution after intranasal administration. RESULTS AND DISCUSSION: The RH-CSNPs showed sustained release profiles for up to 18 h. The RH concentrations (% Radioactivity/g) in the brain following intranasal administration (i.n.) of RH-CSNPs were found to be significantly higher at all the time points compared with RH solution. The concentration of RH was highest in the liver (7.210 ± 0.52), followed by kidneys (6.862 ± 0.62), intestine (4.862 ± 0.45), and lungs (4.640 ± 0.92) in rats following i.n. administration of RH-CSNPs. Gamma scintigraphy imaging in rats was performed to ascertain the localization of drug in the brain following intranasal administration of formulations. The brain/blood ratios obtained (0.251 ± 0.09 and 0.386 ± 0.57 of RH (i.n.) and RH-CSNPs (i.n.), respectively) at 0.5 h are indicative of direct nose to brain transport, bypassing the blood-brain barrier (BBB). CONCLUSION: The novel formulation showed the superiority of nose to brain delivery of RH using mucoadhesive nanoparticles compared with other delivery routes reported earlier.

Preparation, characterization, in vivo biodistribution and pharmacokinetic studies of donepezil-loaded PLGA nanoparticles for brain targeting.

OBJECTIVE: Alzheimer's disease (AD) is a progressive neurodegenerative disorder manifested by cognitive, memory deterioration and variety of neuropsychiatric symptoms. Donepezil is a reversible cholinesterase inhibitor used for the treatment of AD. The purpose of this work is to prepare a nanoparticulate drug delivery system of donepezil using poly(lactic-co-glycolic acid) (PLGA) for sustained release and efficient brain targeting. MATERIALS AND METHODS: PLGA nanoparticles (NPs) were prepared by the solvent emulsification diffusion-evaporation technique and characterized for particle size, particle-size distribution, zeta potential, entrapment efficiency, drug loading and interaction studies and in vivo studies using gamma scintigraphy techniques. RESULTS AND DISCUSSION: The size of drug-loaded NPs (drug polymer ratio 1:1) was found to be 89.67 ± 6.43 nm. The TEM and SEM images of the formulation suggested that particle size was within 20-100 nm and spherical in shape, smooth morphology and coating of Tween-80 on the NPs was clearly observed. The release behavior of donepezil exhibited a biphasic pattern characterized by an initial burst release followed by a slower and continuous sustained release. The biodistribution studies of donepezil-loaded PLGA NPs and drug solution via intravenous route revealed higher percentage of radioactivity per gram in the brain for the nanoparticulate formulation as compared with the drug solution (p < 0.05). CONCLUSION: The high concentrations of donepezil uptake in brain due to coated NPs may help in a significant improvement for treating AD. But further, more extensive clinical studies are needed to check and confirm the efficacy of the prepared drug delivery system.

Lipid drug conjugate nanoparticle as a novel lipid nanocarrier for the oral delivery of decitabine: ex vivo gut permeation studies.

The purpose of this study was to develop lipid drug conjugate (LDC) nanoparticles of decitabine (DCB) using stearic acid as a lipid to increase the permeability of the drug along with its protection from chemical degradation. The LDC was prepared by salt formation of DCB with stearic acid and followed by cold homogenization technique to produce the LDC nanoparticles. The role of key independent variables influencing on dependent variables were determined by using a Box-Behnken design. The optimized batch revealed spherical morphology under TEM analysis with particle size of 202.6 ± 1.65 nm and 0.334 ± 0.987 PDI. The zeta potential and %EE were found to be -33.6 ± 0.845 mV and 68.89% ± 0.59 respectively. Lyophilized powder showed the crystalline structure under DSC analysis. In vitro release studies showed the initial burst release followed by a sustained release up to 24 h in PBS pH 7.4 and the data were further studied using release kinetic models which revealed the first-order model as a best-fitting model. Ex vivo gut permeation studies proved that the formulation containing lipid and surfactants has a higher permeability than the plain drug solution with nearly fourfold increase in the apparent permeability coefficients. Finally, LDC nanoparticles prepared by using stearic acid as a lipid and surfactants as Tween 80, Poloxamer 188, and Labrasol in equal ratio possess high potential for the oral delivery of hydrophilic drugs.

Reduced bioavailability of tamoxifen and its metabolite 4-hydroxytamoxifen after oral administration with biochanin A (an isoflavone) in rats.

The aim of this study was to investigate the effect of biochanin A (BCA) on the pharmacokinetics of tamoxifen, a substrate of P-glycoprotein (P-gp) and cytochrome 3A (CYP3A), in female rats. The tamoxifen was administered orally (10 mg/kg) without or with oral BCA (100 mg/kg) in female rats. As BCA is an inhibitor of CYP 3A and P-gp it was expected to increase the bioavailability of tamoxifen, a known substrate of CYP3A4/Pgp. Surprisingly, compared with the control group (treated with tamoxifen alone), BCA pretreated animals showed significantly (p < 0.05) decreased area under the plasma concentration-time curve from time zero to time infinity (AUC(0-∞)) and peak tamoxifen concentrations (C(max)). Consequently, the relative bioavailability (RB%) of tamoxifen co-administered with BCA was remarkably decreased compared with the control group. The AUC(0-∞) and C(max) of 4-hydroxytamoxifen in BCA pretreated rats were also significantly (p < 0.05) lower than those from the control group. However, there were no apparent changes in the metabolite ratio (MR; AUC(0-∞) of 4-hydroxytamoxifen to tamoxifen) by co-administration of BCA. If the results of this study are further confirmed by clinical trials, tamoxifen dosages should be adjusted to avoid potential drug interaction when tamoxifen is used clinically in combination with BCA and BCA-containing dietary supplements.

Reassessing bioavailability of silymarin.

Silymarin, a flavonolignan derived from Silybum marianum, possesses diverse pharmacological activities, including hepatoprotective, antioxidant, anti-inflammatory, anticancer, and cardioprotective. Although clinical trials have shown silymarin is safe at high doses (>1500 mg/day) in humans, the pharmacokinetic studies over the past three decades related to absorption, distribution, metabolism, and excretion of silymarin have revealed poor absorption, rapid metabolism, and ultimately poor oral bioavailability. For optimum silymarin bioavailability, issues of solubility, permeability, metabolism, and excretion must be addressed. An array of methods have been described in recent years that can improve its bioavailability, including complexation with ß-cyclodextrins, solid dispersion method, formation of microparticles and nanoparticles, self-microemulsifying drug delivery systems, micelles, liposomes, and phytosomes. This article critically reviews the recent published literature on various techniques for increasing the bioavailability of silymarin.

High-throughput quantification of isoflavones, biochanin A and genistein, and their conjugates in female rat plasma using LC-ESI-MS/MS: Application in pharmacokinetic study.

Isoflavones containing foods and dietary supplements are widely consumed for putative health benefits (e.g. cancer chemoprevention, beneficial effects on serum lipids associated with cardiovascular health, reduction of osteoporosis, relief of menopausal symptoms). This paper describes the development and validation of a sensitive high throughput LC-ESI-MS/MS method for quantifying biochanin A (BCA) and genistein (GEN), and their conjugates in rat plasma. The analytes were separated on a Supelco Discovery C18 (4.6×50 mm, 5.0 µm) column under isocratic condition using acetonitrile/methanol (50:50, v/v) and 0.1% acetic acid in the ratio of 90:10 v/v as a mobile phase. The intra- and inter-day assay precision ranged from 2.66 to 8.34% and 4.40 to 8.10% (RSD %), respectively, and intra- and inter-day assay accuracy was between 90.67-109.25% and 95.86-106.32%, respectively, for both the analytes. The lowest quantitation limit for BCA and GEN was 0.5 ng/mL in 0.1 mL of rat plasma. The method was successfully applied to the estimation of BCA, GEN and their conjugates in rat plasma following oral administration of BCA. Circulating conjugates (glucuronides/sulfates) of BCA and GEN were quantified using enzymatic hydrolysis of plasma samples. The levels of isoflavones glucuronides/sulfates were found to be much greater than the corresponding aglycones.

Novel buccal adhesive system for anti-hypertensive agent nimodipine.

Novel buccal adhesive system (NBAS) containing Nimodipine (N) was prepared and evaluated by different parameters such as weight uniformity, thickness, hardness, surface pH, swelling index, mucoadhesive strength (MS), in vitro drug release and ex vivo drug permeation. Different formulations containing 5-20% Carbopol 934 (CP) and SCMC HV in sustained release part and sodium alginate:chitosan lactate in different ratios (1:1, 2:1, 1:2, and 3:1) in fast release part were prepared and tested. NBAS containing CP 5% and SCMC HV and sodium alginate: chitosan lactate 1:1 was selected as a suitable formula based on the (MS) and the release profile. Compared to the conventional buccal adhesive tablet, NBAS showed an effective controlled release pattern with faster release at the initial period. The mechanism of N release was found to be by non-fickian diffusion, followed first order release kinetics. It can be considered that NBAS is a superior, novel system that overcomes the drawback associated with the conventional buccal adhesive tablet.

Solid dispersion as an approach for bioavailability enhancement of poorly water-soluble drug ritonavir.

Ritonavir is an antiretroviral drug characterized by low solubility and high permeability which corresponds to BCS class II drug. The purpose of the study was to develop solid dispersion by different methods and investigate them for in vitro and in vivo performance for enhancing dissolution and bioavailability, respectively. Since the drug possesses food-related absorption, the effect of biorelevant media (FaSSIF and FeSSIF state) on dissolution behavior was also studied. The solid dispersion was prepared using Gelucire as carrier in 1:4 ratio by different methods and were characterized for differential scanning calorimetry (DSC), X-ray diffractometry, scanning electron microscopy, and FT-IR. Oral bioavailability of 10 mg of ritonavir in solid dispersion prepared by solvent evaporation (SE1) and melt method (MM1) was compared with pure drug after oral administration of solid dispersion and pure drug to Albino Wistar rats of either sex. The results suggested formation of eutectic solid dispersion. In vitro dissolution studies was performed in 0.1 N HCl and biorelevant media showed enhanced dissolution rate as compared to pure drug in both FeSSIF media and 0.1 N HCl. The apparent rate of absorption of ritonavir from SE1 (C(max) 20221.37 ng/ml, t(max) 0.5 h) was higher than that of MM1 (C(max) 2,462.2, t(max) 1 h) and pure drug (C(max) 1,354.8 ng/ml, t(max) 0.5 h). On the basis of the result obtained, it was concluded that solid dispersion is a good approach to enhance solubility and bioavailability of poorly water-soluble ritonavir.

Development and evaluation of buccal bioadhesive tablet of an anti-emetic agent ondansetron.

The aim of the present study was to develop and evaluate a buccal adhesive tablet containing ondansetron hydrochloride (OH). Special punches and dies were fabricated and used while preparing buccal adhesive tablets. The tablets were prepared using carbopol (CP 934), sodium alginate, sodium carboxymethylcellulose low viscosity (SCMC LV), and hydroxypropylmethylcellulose (HPMC 15cps) as mucoadhsive polymers to impart mucoadhesion and ethyl cellulose to act as an impermeable backing layer. The formulations were prepared by direct compression and characterized by different parameters such as weight uniformity, content uniformity, thickness, hardness, swelling index, in vitro drug release studies, mucoadhesive strength, and ex vivo permeation study. As compared with the optimized formulation composed of OH--5 mg, CP 934--30 mg, SCMC LV--165 mg, PEG 6000--40 mg, lactose--5 mg, magnesium stearate--1.5 mg, and aspartame--2 mg, which gave the maximum release (88.15%), non-bitter (OH) that form namely ondansetron base and complexed ondansetron was used in order to make the selected formulation acceptable to human. The result of the in vitro release studies and permeation studies through bovine buccal mucosa revealed that complexed ondansetron gave the maximum release and permeation. The stability of drug in the optimized adhesive tablet was tested for 6 h in natural human saliva; both the drug and device were found to be stable in natural human saliva. Thus, buccal adhesive tablet of ondansetron could be an alternative route to bypass the hepatic first-pass metabolism and to improve the bioavailability of (OH).

In-vivo evaluation in rats of colon-specific microspheres containing 5-fluorouracil.

The aims of this investigation were to determine the distribution in the gastrointestinal (GI) tract of Eudragit S-100 encapsulated colon-specific sodium alginate microspheres containing 5-fluorouracil (5-FU) in rats, and to perform pharmacokinetic and pharmacodynamic studies. Comparisons were with a control immediate-release (IR) formulation of 5-FU. 5-FU was distributed predominantly in the upper GI tract from the IR formulation but was distributed primarily to the lower part of the GI tract from the microsphere formulation. No drug was released in the stomach and intestinal regions from the colon-specific microspheres. Significantly, a high concentration of the active drug was achieved in colonic tissues from the colon-specific microspheres (P < 0.001), which was higher than the IC50 required to halt the growth of and/or kill colon cancer cells. Colon cancer was induced in rats by subcutaneous injection of 1,2-dimethylhydrazine (40 mg kg (-1)) for 10 weeks. The tumours induced were non-invasive adenocarcinomas and were in Duke's stage A. The 5-FU formulations were administered for 4 weeks after tumour induction. Non-significant reductions in tumour volume and multiplicity were observed in animals given the colon-specific microspheres. Enhanced levels of liver enzymes (SGOT, SGPT and alkaline phosphatase) were found in animals given the IR formulation of 5-FU, and values differed significantly (P < 0.001) from those in animals treated with the colon-specific microspheres. Elevated levels of serum albumin and creatinine, and leucocytopenia and thrombocytopenia were observed in the animals given the IR formulation. In summary, Eudragit S-100 coated alginate microspheres delivered 5-FU to colonic tissues, with reduced systemic side-effects. A long-term dosing study is required to ascertain the therapeutic benefits.

Transdermal delivery of valsartan: I. Effect of various terpenes.

The objective of the present study was to investigate the effect of various terpenes, including a diterpene, forskolin (FSK; a putative penetration enhancer), on skin permeation of valsartan. Permeation studies were carried out with Automated Transdermal Diffusion Cells Sampling System (SFDC 6, LOGAN Instruments Corp., NJ, USA) through rat skin and human cadaver skin (HCS) using ethanol: IPB (pH 7.4) (40:60) as vehicle. The efficacy of the study terpenes for permeation of valsartan across rat skin and human cadaver skin was found in the order of cineole > d-limonene > l-menthol > linalool > FSK and cineole > d-limonene > linalool > l-menthol > FSK, respectively. No apparent skin irritation (erythema, edema) was observed on treatment of skin with terpenes including FSK. FT-IR, DSC, and histopathological studies revealed that FSK enhanced the skin permeation of the active drug by disruption and extraction of lipid bilayers of SC in consonance with other terpenes.

Development and in vitro evaluation of an acid buffering bioadhesive vaginal gel for mixed vaginal infections.

An acid buffering bioadhesive vaginal (ABBV) gel was developed for the treatment of mixed vaginal infections. Different bioadhesive polymers were evaluated on the basis of their bioadhesive strength, stability and drug release properties. Bioadhesion and release studies showed that guar gum, xanthan gum and hydroxypropyl methylcelullose K4M formed a good combination of bioadhesive polymers to develop the ABBV gel. Monosodium citrate was used as an acid buffering agent to provide acidic pH (4.4). The drugs clotrimazole (antifungal) and metronidazole (antiprotozoal as well as antibacterial) were used in the formulation along with Lactobacillus spores to treat mixed vaginal infections. The ex vivo retention study showed that the bioadhesive polymers hold the gel for 12-13 hours inside the vaginal tube. Results of the in vitro antimicrobial study indicated that the ABBV gel had better antimicrobial action than the commercial intravaginal drug delivery systems and retention was prolonged in an ex vivo retention experiment.

Development and bioavailability assessment of ramipril nanoemulsion formulation.

The objective of our investigation was to design a thermodynamically stable and dilutable nanoemulsion formulation of Ramipril, with minimum surfactant concentration that could improve its solubility, stability and oral bioavailability. Formulations were taken from the o/w nanoemulsion region of phase diagrams, which were subjected to thermodynamic stability and dispersibility tests. The composition of optimized formulation was Sefsol 218 (20% w/w), Tween 80 (18% w/w), Carbitol (18% w/w) and standard buffer solution pH 5 (44% w/w) as oil, surfactant, cosurfactant and aqueous phase, respectively, containing 5 mg of ramipril showing drug release (95%), droplet size (80.9 nm), polydispersity (0.271), viscosity (10.68 cP), and infinite dilution capability. In vitro drug release of the nanoemulsion formulations was highly significant (p<0.01) as compared to marketed capsule formulation and drug suspension. The relative bioavailability of ramipril nanoemulsion to that of conventional capsule form was found to be 229.62% whereas to that of drug suspension was 539.49%. The present study revealed that ramipril nanoemulsion could be used as a liquid formulation for pediatric and geriatric patients and can be formulated as self-nanoemulsifying drug delivery system (SNEDDS) as a unit dosage form.

Development and evaluation of acid-buffering bioadhesive vaginal tablet for mixed vaginal infections.

An acid-buffering bioadhesive vaginal tablet was developed for the treatment of genitourinary tract infections. From the bioadhesion experiment and release studies it was found that polycarbophil and sodium carboxymethylcellulose is a good combination for an acid-buffering bioadhesive vaginal tablet. Sodium monocitrate was used as a buffering agent to provide acidic pH (4.4), which is an attribute of a healthy vagina. The effervescent mixture (citric acid and sodium bicarbonate) along with a superdisintegrant (Ac-Di-sol) was used to enhance the swellability of the bioadhesive tablet. The drugs clotrimazole (antifungal) and metronidazole (antiprotozoal as well as an antibacterial) were used in the formulation along with Lactobacillus acidophilus spores to treat mixed vaginal infections. From the ex vivo retention study it was found that the bioadhesive polymers hold the tablet for more than 24 hours inside the vaginal tube. The hardness of the acid-buffering bioadhesive vaginal tablet was optimized, at 4 to 5 kg hardness the swelling was found to be good and the cumulative release profile of the developed tablet was matched with a marketed conventional tablet (Infa-V). The in vitro spreadability of the swelled tablet was comparable to the marketed gel. In the in vitro antimicrobial study it was found that the acid-buffering bioadhesive tablet produces better antimicrobial action than marketed intravaginal drug delivery systems (Infa-V, Candid-V and Canesten 1).

Design and evaluation of bilayer floating tablets of captopril.

The objective of the present investigation was to develop a bilayer-floating tablet (BFT) for captopril using direct compression technology. HPMC, K-grade and effervescent mixture of citric acid and sodium bicarbonate formed the floating layer. The release layer contained captopril and various polymers such as HPMC-K15M, PVP-K30 and Carbopol 934p, alone or in combination with the drug. The floating behavior and in vitro dissolution studies were carried out in a USP 23 apparatus 2 in simulated gastric fluid (without enzyme, pH 1.2). Final formulation released approximately 95% drug in 24 h in vitro, while the floating lag time was 10 min and the tablet remained floatable throughout all studies. Final formulation followed the Higuchi release model and showed no significant change in physical appearance, drug content, floatability or in vitro dissolution pattern after storage at 45 degrees C/75% RH for three months. Placebo formulation containing barium sulphate in the release layer administered to human volunteers for in vivo X-ray studies showed that BFT had significantly increased the gastric residence time.

Characterization of 5-fluorouracil microspheres for colonic delivery.

The purpose of this investigation was to prepare and evaluate the colon-specific microspheres of 5-fluorouracil for the treatment of colon cancer. Core microspheres of alginate were prepared by the modified emulsification method in liquid paraffin and by cross-linking with calcium chloride. The core microspheres were coated with Eudragit S-100 by the solvent evaporation technique to prevent drug release in the stomach and small intestine. The microspheres were characterized by shape, size, surface morphology, size distribution, incorporation efficiency, and in vitro drug release studies. The outer surfaces of the core and coated microspheres, which were spherical in shape, were rough and smooth, respectively. The size of the core microspheres ranged from 22 to 55 microm, and the size of the coated microspheres ranged from 103 to 185 microm. The core microspheres sustained the drug release for 10 hours. The release studies of coated microspheres were performed in a pH progression medium mimicking the conditions of the gastrointestinal tract. Release was sustained for up to 20 hours in formulations with core microspheres to a Eudragit S-100 coat ratio of 1:7, and there were no changes in the size, shape, drug content, differential scanning calorimetry thermogram, and in vitro drug release after storage at 40 degrees C/75% relative humidity for 6 months.

Formulation, development and optimization of raloxifene-loaded chitosan nanoparticles for treatment of osteoporosis.

CONTEXT: Osteoporosis (OP) is a disease of skeletal system and is associated with fragility fracture at the hip, spine and wrist. Various drugs have been used to treat OP. One of them is raloxifene hydrochloride (RLX), a second-generation selective estrogen receptor modulator (SERM) approved by the USFDA. RLX possesses only 2% absolute bioavailability (BA) by oral route due to its extensive first-pass metabolism. OBJECTIVE: The purpose of the current research work was to develop and evaluate RLX-loaded chitosan nanoparticles (CS-NPs) for treatment of OP with enhanced BA. MATERIALS AND METHODS: The RLX-loaded CS-NPs were prepared by gelation of CS with tripolyphosphate (TPP) by ionic cross-linking. Formulation was optimized and in vitro drug release and in vivo study were performed. RESULTS AND DISCUSSIONS: CS-NPs were formed by the ionic gelation method. The particle size, entrapment efficiency and loading efficiency varied from 216.65 to 1890 nm, 32.84 to 97.78% and 23.89 to 62.46%, respectively. Release kinetics showed diffusion-controlled and Fickian release pattern. In vivo study indicated higher plasma drug concentration with NPs administered intranasally as compared to drug suspension administered through oral route (p < 0.05). A significantly higher drug concentration in plasma was achieved in 10 min after nasal administration with respect to oral administration. CONCLUSION: The results suggest that RLX-loaded CS-NPs have better BA and would be a promising approach for intranasal (i.n.) delivery of RLX for the treatment of OP.

Nanoethosomes mediated transdermal delivery of vinpocetine for management of Alzheimer's disease.

AIM: To develop and statistically optimize nanoethosomal formulation for transdermal delivery of vinpocetine as an anti-Alzheimer's drug. MATERIALS AND METHODS: Box-Behnken experimental design was applied for optimization of nanoethosomes. The independent variables were phospholipid (X1), Tween 80 (X2) and Ethanol (X3) while entrapment efficiency (Y1), particle sizes (Y2), elasticity (Y3) and flux (Y4) were the dependent variables. RESULTS AND CONCLUSION: Optimized nanoethosomal vinpocetine formulation with mean particle size 50.57 ± 26.11 nm showed 97.51 ± 0.86% entrapment efficiency, achieved mean transdermal flux 925.60 ± 39.80 µg/cm2/h and elasticity of 86.61 ± 2.88. Ex-vivo study of nanoethosomal formulation showed a significant increase flux and entrapment efficiency compared with control vinpocetine solution. Our results suggest that nanoethosome is an efficient carrier for transdermal delivery of vinpocetine as compared to its oral form.

Improved immune response from biodegradable polymer particles entrapping tetanus toxoid by use of different immunization protocol and adjuvants.

Poly lactide-co-glycolide (PLGA) and polylactide (PLA) particles entrapping immunoreactive tetanus toxoid (TT) were prepared using the solvent evaporation method. The effect of different formulation parameters such as polymer hydrophobicity, particle size and use of additional adjuvants on the generation of immune responses in experimental animals was evaluated. Immune responses from hydrophobic polymer particles were better than those from hydrophilic polymer. Immunization with physical mixtures of different size particles resulted in further improvement in anti-TT antibody titers in Wistar rats. Physical mixture of nano and microparticles resulted in early as well as high antibody titers in experimental animals. Immunization with polymer particles encapsulating stabilized TT elicited anti-TT antibody titers, which persisted for more than 5 months and were higher than those obtained with saline TT. However, antibody responses generated by single point immunization of either particles or physical mixture of particles were lower than the conventional two doses of alum-adsorbed TT. Immunization with nanoparticles along with alum resulted in very high and early immune response: high anti-TT antibody titers were detected as early as 15 days post-immunization. Use of a squalene emulsion along with the particles during immunization enhanced the level of anti-TT antibody titers considerably. Single point immunization with admixtures of PLA microparticles and alum resulted in antibody response very close to that achieved by two injections of alum-adsorbed TT; the antibody titers were more than 50 microg/ml over a period of 6 months. These results indicated that the judicious choice of polymer and particles size, protecting the immunoreactivity of the entrapped antigen and the appropriate design of immunization protocol along with suitable adjuvant can lead to the generation of long lasting immune response from single dose vaccine formulation using polymer particles.

Potentiation of immune response from polymer-entrapped antigen: toward development of single dose tetanus toxoid vaccine.

Poly(lactide) (PLA) polymer particles entrapping immunoreactive tetanus toxoid (TT) were used for generation of immune response using single point immunization. Immunization with different sizes of polymer particles encapsulating immunoreactive TT elicited anti-TT antibody titers that persisted for more than 5 months. However, antibody response generated by single point immunization of either nanoparticles or microparticles were lower than the conventional two doses of alum adsorbed TT. To overcome this limitation, alum was used with particles that improved anti-TT antibody response. Immunization with nanoparticles along with alum resulted in very high and early immune response: high anti-TT antibody titers were detected as early as 15 days postimmunization. However anti-TT antibody titers declined rapidly with time. Immunization with admixture of microparticles and alum elicited higher antibody titers than the particles alone and the antibody titers were high particularly during the later part of the postimmunization period. Single point immunization with admixture of PLA microparticles and alum resulted in an antibody response very close to that achieved by two injection of alum-adsorbed TT. Physical mixture of both a nano- and microparticles along with alum resulted in sustained anti-TT antibody response from very early days of postimmunization until 150 days. The antibody titers were maintained around 50 microg/ml for more than 5 months. These results indicated that immune response from polymer particles can be further improved by use of additional adjuvant. Furthermore, using various size particles or physical mixture of different size particles along with alum, it is possible to modulate the kinetics of immune response using polymer particles based immunization.