Effect of Eudragit on In Vitro Transfection Efficiency of PEI-DNA Complexes.

AIM: Eudragit® E 100 (EE100) was used to improve the transfection efficiency of polyethylenimine (PEI). MATERIALS AND METHODS: Mobility of PEI-DNA complexes with and without EE100 were visualized by agarose gel electrophoresis and their transfection efficiencies were investigated in KB human oral carcinoma cells by flow cytometry. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay was used to determine the viability of transfected cells. RESULTS: Gel electrophoresis illustrated formation of complete complexes at N/P ratios above 5. PEI had the highest transfection efficiency at an N/P ratio of 15, whereas in combination with EE100, the transfection efficiency was highest at an N/P ratio of 7.5. High concentrations of EE100 in combination with PEI were found to reduce cell viability. CONCLUSION: The results show a synergistic action of EE100 in transfection of DNA at low N/P ratios compared to PEI alone.

Enhanced bioavailability of danazol nanosuspensions by wet milling and high-pressure homogenization.

INTRODUCTION: The majority of drugs obtained through synthesis and development show poor aqueous solubility and dissolution velocity, resulting in reduced bioavailability of drugs. Most of these problems arise from formulation-related performance issues, and an efficient way to overcome these obstacles and to increase dissolution velocity is to reduce the particle size of drug substances to form drug nanosuspensions. MATERIALS AND METHODS: Danazol nanosuspensions were prepared by wet milling (WM) and high-pressure homogenization (HPH) methods. The nanosuspensions obtained using these fabrication methods were analyzed for their particle size, surface charge, and the crystallinity of the product was assessed by X-ray diffraction (XRD) and differential scanning calorimetry techniques. To determine in vitro and in vivo performances of the prepared nanosuspensions, dissolution velocity, and bioavailability studies were performed. RESULTS: Particle size and zeta potential analysis showed the formation of nanosized particles with a negative charge on the surface. XRD depicted the nanocrystalline nature of danazol with low diffraction intensities. With increased surface area and saturation solubility, the nanosuspensions showed enhanced dissolution velocity and oral bioavailability in rats when compared to the bulk danazol suspension. CONCLUSIONS: The results suggest that the preparation of nanosuspensions by WM or HPH is a promising approach to formulate new drugs or to reformulate existing drugs with poorly water-soluble properties.

Treatment of experimental autoimmune encephalomyelitis by codelivery of disease associated Peptide and dexamethasone in acetalated dextran microparticles.

Multiple sclerosis (MS) is an autoimmune, demyelinating disease of the central nervous system that can cause loss of motor function and is thought to result, in part, from chronic inflammation due to an antigen-specific T cell immune response. Current treatments suppress the immune system without antigen specificity, increasing the risks of cancer, chronic infection, and other long-term side effects. In this study, we show treatment of experimental autoimmune encephalomyelitis (EAE), a model of MS, by coencapsulating the immunodominant peptide of myelin oligodendrocyte glycoprotein (MOG) with dexamethasone (DXM) into acetalated dextran (Ac-DEX) microparticles (DXM/MOG/MPs) and administering the microparticles subcutaneously. The clinical score of the mice was reduced from 3.4 to 1.6 after 3 injections 3 days apart with the coencapsulated microparticulate formulation (MOG 17.6 µg and DXM 8 µg). This change in clinical score was significantly greater than observed with phosphate-buffered saline (PBS), empty MPs, free DXM and MOG, DXM/MPs, and MOG/MPs. Additionally, treatment with DXM/MOG/MPs significantly inhibited disease-associated cytokine (e.g., IL-17, GM-CSF) expression in splenocytes isolated in treated mice. Here we show a promising approach for the therapeutic treatment of MS using a polymer-based microparticle delivery platform.

Enhanced stability of horseradish peroxidase encapsulated in acetalated dextran microparticles stored outside cold chain conditions.

Micro- and nanoparticles have been shown to improve the efficacy of safer protein-based (subunit) vaccines. Here, we evaluate a method of improving the vaccine stability outside cold chain conditions by encapsulation of a model enzyme, horseradish peroxidase (HRP), in an acid-sensitive, tunable biodegradable polymer, acetalated dextran (Ac-DEX). Vaccines that are stable outside the cold chain would be desirable for use in developing nations. Ac-DEX particles encapsulating HRP were prepared using two different methods, probe sonication and homogenization. These particles were stored under different storage conditions (-20 °C, 4 °C, 25 °C or 45 °C) for a period of 3 months. On different days, the particles were characterized for various physical and chemical measurements. At all conditions, Ac-DEX particles remained spherical in nature, as compared to PLGA particles that fused together starting at day 3 at 45 °C. Furthermore, our results indicated that encapsulation of HRP in Ac-DEX reduces its storage temperature dependence and enhances its stability outside cold chain conditions. Homogenized particles performed better than probe sonicated particles and retained 70% of the enzyme's initial activity as compared to free HRP that retained only 40% of the initial activity after 3 months of storage at 25 °C or 45 °C. Additionally, HRP activity was more stable when encapsulated in Ac-DEX, and the variance in enzyme activity between the different storage temperatures was not observed for either particle preparation. This suggests that storage at a constant temperature is not required with vaccines encapsulated in Ac-DEX particles. Overall, our results suggest that an Ac-DEX based micro-/nanoparticles system has wide applications as vaccines and drug delivery carriers, including those in developing nations.

Optimization of rapamycin-loaded acetalated dextran microparticles for immunosuppression.

Immunosuppressive drugs can treat autoimmune disorders and limit rejection with organ transplants. However, delivering immunosuppressants like rapamycin systemically can have harmful side-effects. We aim to potentially reduce these toxic side-effects by encapsulating rapamycin in a polymeric microparticle to passively target phagocytes, the cells integral in immunosuppression. Acetalated dextran (Ac-DEX) is a recently described, biocompatible polymer which undergoes tunable burst degradation at the acidic conditions present in the phagosome (pH 5) but slower degradation at extracellular conditions (pH 7.4), thereby making it an ideal candidate for immune applications. Rapamycin-loaded microparticles were fabricated from Ac-DEX through a single emulsion (water/oil) technique. Optimized microparticles were determined by varying the chemical and physical parameters during particle synthesis. Microparticles synthesized from Ac-DEX with a molecular weight of 71 k had higher encapsulation efficiency of rapamycin and slower overall degradation than microparticles synthesized from 10k Ac-DEX. To evaluate the ability of rapamycin-loaded Ac-DEX microparticles to reduce a pro-inflammatory response, they were incubated with lipopolysaccharide-stimulated RAW macrophages. RAW macrophages treated with rapamycin-loaded microparticles exhibited reduced nitric oxide production and favorable cell viability. Overall, we have shown optimization of immunosuppressive rapamycin-loaded microparticles using the novel polymer Ac-DEX. These particles will be advantageous for future applications in immune suppression therapies.

SPANosomes as delivery vehicles for small interfering RNA (siRNA).

Nonionic surfactant vesicles, or SPANosomes (SPs), comprised of cationic lipid and sorbitan monooleate (Span 80) were synthesized and evaluated as small interfering RNA (siRNA) vectors. The SPs had a mean diameter of less than 100 nm and exhibited excellent colloidal stability. The SP/siRNA complexes possessed a slightly positive zeta potential of 12 mV and demonstrated a high siRNA incorporation efficiency of greater than 80%. Cryogenic transmission electron microscopy (cryo-TEM) imaging of the SP/siRNA indicated a predominantly core-shell structure. The SP/siRNA complexes were shown to efficiently and specifically silence expression of both green fluorescent protein (GFP) (66% knockdown) and aromatase (77% knockdown) genes in breast cancer cell lines. In addition, the cellular trafficking pathway of the SP/siRNA was investigated by confocal microscopy using molecular beacons as probes for cytosolic delivery. The results showed efficient endosomal escape and cytosolic delivery of the siRNA cargo following internalization of the SP/siRNA complexes. In conclusion, Span 80 is a potent helper lipid, and the SPs are promising vehicles for siRNA delivery.

Synthesis, optimization, and characterization of camptothecin-loaded acetalated dextran porous microparticles for pulmonary delivery.

We propose the use of a new biopolymer, acetalated dextran (Ac-DEX), to synthesize porous microparticles for pulmonary drug delivery. Ac-DEX is derived from the polysaccharide dextran and, unlike polyesters, has tunable degradation from days to months and pH neutral degradation products. Ac-DEX microparticles fabricated through emulsion techniques were optimized using a variety of postprocessing techniques to enhance the respirable fraction for pulmonary delivery. Tangential flow filtration resulted in a maximum 37% respirable fraction for Ac-DEX porous microparticles, compared to a 10% respirable fraction for poly(lactic-co-glycolic acid) (PLGA) porous microparticles. Ac-DEX microparticles were of an optimum diameter to minimize macrophage clearance but had a low enough theoretical density for deep lung penetration. Transepithelial electrical resistance (TEER) measurements showed that the particles did not impinge on a monolayer of lung epithelial cells in either air or liquid conditions. Also, the release of the chemotherapeutic camptothecin was shown to be tunable depending on Ac-DEX degradation time and molecular weight, and drug release was shown to be bioactive over a range of concentrations. Our results indicate that both release kinetics and fraction of burst release of drug from Ac-DEX porous microparticles can be tuned by simply changing the Ac-DEX polymer properties, affording a large range of formulation options for drug delivery to the pulmonary cavity. Overall, Ac-DEX porous microparticles show promise as an emerging carrier for pulmonary delivery of drugs to the alveolar region of the lung, particularly for the treatment of lung diseases.

Nanoparticulate delivery of novel drug combination regimens for the chemoprevention of colon cancer.

The purpose of this work was to assess synergistic inhibitory responses of a novel chemopreventive combination regimen of drugs namely, aspirin in combination with calcium and folic acid on two human colon cancer cell lines, HT-29 and SW-480. Subsequently, based on positive responses, nanotechnology-based formulations were developed for the targeted delivery of these combinatorial regimens to the colon for the chemoprevention of colon cancer. Additionally, conventional drug formulations using controlled release polymers chitosan, pectin and hydroxypropyl methylcellulose (HPMC) were tested for release of the drugs, for comparison purposes. Chemopreventive combination regimens demonstrated significant synergistic efficacy in both cell lines from XTT assay studies, when compared to the effects of individual agents. Approximately 45% decrease in cell viability for aspirin (15 mM) and calcium (30 mM) mixtures was observed in HT-29 cell lines, compared to approximately 55% decrease by the same combination in SW-480 cell lines. With combinations of aspirin (5 mM) and folic acid (1.5 mM), HT-29 cells demonstrated a 30% decrease in cell viability compared to approximately 38% decrease in the SW-480 cell line. Overall, all drug combinations demonstrated significant synergistic responses in the cell lines tested with the SW-480 cell line being more significantly affected by the drug regimens than the HT-29 cell line. Drug encapsulated nanoparticles demonstrated a spherical morphology, <125 nm average particle size (aspirin and folic acid) of nanoparticles and encapsulation efficiencies in the range of 80-91%. Drug release from nanoparticles was controlled with approximately 60% of the original amount released over a 96 h period. Conventional formulations exhibited faster kinetics of drug release when compared to the PLGA nanoparticles. Overall, the cell line studies demonstrate, for the first time, the ability of novel chemopreventive combinations to inhibit the growth of colon cancer cells whereas the nanotechnology-based drug delivery system provides valuable evidence for targeted therapy towards colon cancer chemoprevention.

Novel combinations of rate-controlling polymers for the release of leuprolide acetate in the colon.

Our objective was to investigate the controlled release and transport of leuprolide acetate polypeptide in the colon using novel combinations of rate-controlling polymers. Polymer swelling, disintegration, drug release, and transport characteristics were measured using different polymers, carbopol, chitosan and polyox, alone and in combination. Studies demonstrated Carbopol-containing combination formulations had maximum swelling and the slowest disintegration properties. A decrease in dissolution rate was observed from all combination formulations when compared with their individual counterparts. Carbopol combinations showed the slowest overall release. Drug transport studies using the everted sac technique demonstrated good correlation to the swelling, disintegration, and dissolution studies. Thus, novel polymer combinations can be used to deliver polypeptide drug to the colon effectively compared with individual polymers.