Abstract: Cells of the mononuclear phagocytic system, in particular monocytes/macrophages (Mo/Mac) serve as a reservoir for human immunodeficiency virus (HIV) and are believed to be responsible for its dissemination throughout the body and especially into the brain. Treatment of HIV infection, therefore, must reach these cells in addition to the lymphocytes. The purpose of the present study is to develop poly(propyleneimine) (PPI) dendrimer-based nanocontainers for targeting of efavirenz (EFV) to Mo/Mac. Fifth generation PPI dendrimer, t-Boc–glycine conjugated PPI dendrimer (TPPI) and mannose conjugated dendrimers were synthesized and characterized. While the haemolytic activity and cytotoxicity of PPI dendrimer was found to be very high, the toxicity of t-Boc–glycine conjugated dendrimer and mannose conjugated dendrimers were found to be negligible. The entrapment efficiency of mannose conjugated dendrimer was found to be 47.4%, followed by that of PPI dendrimer (32.15%) and t-Boc–glycine conjugated dendrimer (23.1%). The in vitro drug release profile shows that while PPI dendrimer releases the drug by 24 h, the dendrimer-based nanocontainers prolong the release rate up to 144 h (83 ^ 0.4% in case of t-Boc–glycine conjugated dendrimer and 91 ^ 0.3% in mannose conjugated dendrimer). The cellular uptake of EFV was found to be both concentration and time dependent. Significant increase in cellular uptake of EFV by Mo/Mac cells were observed in case of mannose conjugated dendrimer which is 12 times higher than that of free drug and 5.5 times higher than that of t-Boc–glycine conjugated dendrimer. While mannose conjugated dendrimer was taken up by the lectin receptors of the cells, phagocytosis of t-Boc–glycine conjugated dendrimer might be responsible for its enhanced uptake. Results suggest that the proposed carriers hold potential to increase the efficacy and reduce the toxicity of antiretroviral therapy.

Abstract: Dendrimers are three dimensional polymers, nanoscopic in size, most widely explored in the field of drug delivery in recent times. In order to establish these polymers as controlled and targeted drug delivery systems, they should be non-toxic, biocompatible and biodegradable. The purpose of the present study is to investigate the toxicological profile of fifth generation poly (propyleneimine) dendrimers (PPI) and some of its surface engineered derivatives. Functionalized PPI dendrimers (TPPI, MPPI and TuPPI) were synthesized to mask the primary amino groups responsible for the positive charge and associated toxicity. Each polymer is administered in three different doses viz. 2.5 mg/kg, 25 mg/kg and 250 mg/kg (i.e. low, intermediate and high dose) to Wister rats, and blood as well as tissue samples were collected after 24 h and 15 days. Decrease in RBC count and hemoglobin content after 24 h, in case of animals administered with PPI suggests hemolytic activity of PPI. Significant increase in SGOT, SGPT and LDH indicates that PPI causes severe damage to the membranes of the various tissues of the body, especially that of the liver leading to the leakage of these marker enzymes in blood. Sections of liver of animals administered with PPI showed signs of tissue degeneration after 24 h. No signs of toxicity were observed in case of animals administered with functionalized PPI. Neither PPI nor its surface engineered derivatives showed any signs of immunogenicity. It can be concluded that functionalization of dendrimers leads to drastic reduction of toxicity and increases biocompatibility.

Abstract: The purpose of the present research work is to explore the potential of dendrosomes in genetic immunization against hepatitis B. Plasmid DNA encoding pRc/CMV-HBs(S) (5.6 kb), encoding the small region of the hepatitis B surface antigen, was complexed with 5th generation poly(propyleneimine) dendrimer (PPI) in different ratios. Transfection of CHO cells revealed that a ratio of 1:50 for pDNA: PPI was optimum for transfection. Results of cytotoxicity studies showed that the toxicity of PPI-DNA complex was significantly (p<0.05) higher for PPI 75 and PPI 100 as compared to the other PPI-DNA complexes. PPI 50 was employed for preparation of dendrosomes by reverse phase evaporation method. The dendrosomal formulation DF3 was found to possess optimum vesicle size, zeta potential and entrapment efficiency. In vitro production of HBsAg in CHO cells showed that DF3 possess maximum transfection efficiency. In vivo immunization studies were carried out by giving a single intramuscular injection of 10 μg of plasmid DNA(pDNA) or its dendrimeric or dendrosomal formulation to female Balb/c mice, followed by estimation of total IgG, IgG1, IgG2a, IgG2b, bi weekly. DF3 was found to elicit maximum immune response in terms of total IgG and its subclasses under study as compared to PPI 50 and pDNA at all time points. Animals immunized with DF3 developed very high cytokine level. Higher level of IFN-γ suggests that the immune response was strictly Th1 mediated. Our observations clearly prove the superiority of dendrosomes over PPI-DNA complex and pDNA for genetic immunization against hepatitis B.

Abstract: HIV infected macrophages are considered as reservoirs for spreading the virus in AIDS patients. Tuftsin not only binds specifically to the mononuclear phagocytic cells but also enhances their natural killer activity. The purpose of this study is to explore the targeting potential and anti-HIV activity of Efavirenz (EFV) loaded, tuftsin conjugated 5th generation poly (propyleneimine) dendrimers (TuPPI) in vitro. Tuftsin was chemically conjugated to 5th generation poly (propyleneimine) dendrimers (PPI). The entrapment efficiency of PPI and TuPPI were found to be 37.43 ± 0.3 % and 49.31±0.33% respectively. TuPPI was found to slow down and prolong the in vitro release of EFV upto 144 h against PPI, which releases the drug completely within 24 h. TuPPI possessed negligible cytotoxicity as compared to that of PPI. The cellular uptake of TuPPI was found to be 34.5 times higher than that of the free drug in first 1 h and was significantly higher in HIV infected macrophages than that of uninfected cells. TuPPI was found to reduce the viral load by 99% at a concentration of 0.625 ng/ml, which is due to the enhanced cellular uptake, reduced toxicity and the inherent anti HIV activity of TuPPI.

Abstract: T-lymphocytes, dendritic cells and macrophages are the target cells for HIV. The infected macrophages are considered as reservoirs for spreading the virus. Treatment of HIV infection therefore must reach these cells in addition to the organs like brain, liver and bone marrow. Lectin receptors, which act as molecular targets for sugar molecules, are found on the surface of these cells of the phagocytic system. The purpose of the present study is to investigate the targeting potential and anti HIV activity of lamivudine (3TC) loaded mannosylated fifth generation Poly (propyleneimine) dendrimers (MPPI). The entrapment efficiency of 3TC loaded MPPI and 5th generation poly(propyleneimine) dendrimer (PPI) were found to be 43.27±0.13% and 35.69±0.2% respectively. The in vitro drug release profile shows that while PPI releases the drug by 24 h, the MPPI slows down and hence prolongs the release up to 144 h (96.89±1.8% in case of MPPI). The results of in vitro ligand agglutination assay indicated that even after conjugation with PPI, mannose displayed binding specificity towards Con A. The subtoxic concentrations of free 3TC, blank PPI, blank MPPI, drug loaded PPI and drug loaded MPPI, determined on MT2 cells, were found to be 0.625, 0.039, 0.156, 0.039 and 0.156 nM/ml respectively. Significant increase in cellular uptake of 3TC was observed when MPPI was used, which was 21 and 8.3 times higher than that of free drug (p<0.001) and PPI (p<0.001) at 48 h respectively. Antiretroviral activity was determined using MT2 cell lines by estimating p24 antigen by ELISA. 3TC loaded PPI and MPPI formulations were found to possess higher anti-HIV activity at a concentration as low as 0.019 nM/ml, as compared to that of free drug, which was found to be extremely significant (p<0.001). The significantly higher anti-HIV activity of PPI and MPPI is due to the enhanced cellular uptake of 3TC in formulation as compared to that of free drug Results suggest that the proposed carrier hold potential to increase the efficacy and reduce the toxicity of antiretroviral therapy.

Abstract: Gene transfer to humans requires carriers for the plasmid DNA, which can efficiently and safely carry the gene into the nucleus of the desired cells. The purpose of the present study was to design dendrosomes as a novel, non-viral, vesicular, gene delivery vector and to carry out a comparative study of the relative transfection efficiencies of dendrosomes with standard non-viral, gene delivery vectors. Fourth-generation PAMAM dendrimers were synthesized by double the Michael addition reaction and extensively characterized. The dendrimer–DNA complex was prepared and was confirmed by CD spectroscopy. The dendrosomes were prepared by the reverse phase evaporation method and the entrapment efficiency of the dendrosomal formulation was estimated. In vitro toxicity of the formulation was evaluated by hemolytic toxicity and cytotoxicity studies. Transfection efficiency of the dendrosomal formulations was compared to standard non-viral gene delivery vectors in HEK-293 cell. The results of hemolytic toxicity cytotoxicity studies demonstrated that the dendrosomes possess negligible toxicity as compared to the other formulations and are suitable for in vivo administration. The results of transfection of HEK-293 cell with PGL2 showed that the dendrosomal formulation DF3 possesses superior transfection efficiency against other delivery systems under study. Dendrosomes possess tremendous potential as a novel non-viral and non-toxic gene delivery vector.