Minakshi Garg

Investigations on the toxicological profile of functionalized fifth‐generation poly(propylene imine) dendrimer

Dendrimers have generated tremendous interest in the field of drug delivery. Despite indications of their utility as drug carriers, the inherent cytotoxicity associated with polycationic dendrimers acts as a limiting factor to their clinical applications. Many functionalization strategies have been adopted to mask peripheral amines in order to overcome this limitation. The object of the present investigation was to evaluate the effect of functionalization on the toxicological profile of fifth‐generation poly(propylene imine) dendrimer (PPI‐5.0G). Four forms of functionalized dendrimers, including protected glycine and phenylalanine, and mannose and lactose functionalized poly(propylene imine) (PPI) dendrimer, were synthesized as prospective drug carriers. These dendrimeric systems were evaluated for haemolytic toxicity, cytotoxicity, immunogenicity and haematological parameters. PPI‐5.0G demonstrated a positive charge‐based time‐ and concentration‐dependent toxicity profile. Functionalization greatly improved the toxicity profile of the parent dendrimer. Hence it is proposed that these functionalized forms of PPI dendrimer have great potential as bio‐compatible drug vehicles.

Poly (propyleneimine) dendrimer based nanocontainers for targeting of efavirenz to human monocytes/macrophages in vitro.

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.

Targeting of efavirenz loaded tuftsin conjugated poly(propyleneimine) dendrimers to HIV infected macrophages in vitro

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 144h 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.

Stavudine-loaded mannosylated liposomes : In-vitro anti-HIV-I activity, tissue distribution and pharmacokinetics

Cells of the mononuclear phagocyte system (MPS) are important hosts for human immunodeficiency virus (HIV). Lectin receptors, which act as molecular targets for sugar molecules, are found on the surface of these cells of the MPS. Stavudine‐loaded mannosylated liposomal formulations were developed for targeting to HIV‐infected cells. The mannose‐binding protein concanavalin A was employed as model system for the determination of in‐vitro ligand‐binding capacity. Antiretroviral activity was determined using MT‐2 cell line. Haematological changes, tissue distribution and pharmacokinetic studies of free, liposomal and mannosylated liposomal drug were performed following a bolus intravenous injection in Sprague‐Dawley rats. The entrapment efficiency of mannosylated liposomes was found to be 47.H ± 1.57%. Protein‐carbohydrate interaction has been utilized for the effective delivery of mannosylated formulations. Cellular drug uptake was maximal when mannosylated liposomes were used. MT2 cells treated continuously with uncoated liposomal formulation had p24 levels 8–12 times lower than the level of free drug solution. Further, the mannosylated liposomes have shown p24 levels that were 14–20 and 1.42.3 times lower than the level of free drug and uncoated liposomal formulation treatment, respectively. Similar results were observed when infected MT2 cells were treated overnight. Stavudine, either given plain or incorporated in liposomes, led to development of anaemia and leucocytopenia while mannosylated liposomes overcame these drawbacks. These systems maintained a significant level of stavudine in the liver, spleen and lungs up to 12 h and had greater systemic clearance as compared with free drug or the uncoated liposomal formulation. Mannosylated liposomes have shown potential for the site‐specific and ligand‐directed delivery systems with desired therapeutics and better pharmacological activity.

Reduced hematopoietic toxicity, enhanced cellular uptake and altered pharmacokinetics of azidothymidine loaded galactosylated liposomes

In order to target liposomes to the lectin receptors present on macrophages, galactosylated liposomes were prepared and characterized in vitro. O-palmitoylgalactose (OPG) for liposomal coating was synthesized by esterification of galactose with palmitoyl chloride. The galactose binding Ricinus communis lectin was employed as a model system for the determination of in vitro ligand binding capacity. Cellular drug uptake studies were performed using alveolar macrophages. Hematological changes, bone marrow toxicity, plasma and tissue distribution study of free, uncoated plain liposomal and galactosylated liposomal encapsulated azidothymidine (AZT) were determined following a bolus intravenous injection in Sprague–Dawley rats. Lectin (R. communis) carbohydrate interaction has been utilized for the effective delivery of AZT entrapped in galactosylated vesicles. Aggregation of galactosylated liposomes increased as lectin concentration was increased from 5 to 30 μg/ml. Cellular uptake of galactosylated liposomal formulation was maximum. No hematological toxicity was observed even after 10 days in case of galactosylated vesicle entrapped AZT. This formulation maintained a significant level of AZT in tissues rich in galactose specific receptors and had a prolonged residence in the body resulting in enhanced half-life of AZT. Conclusively, galactosylated liposomes are the potential candidate for targeted drug delivery and are anticipated to be promising in the treatment of AIDS6.

Poly(propyleneimine) dendrimer and dendrosome mediated genetic immunization against hepatitis B

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 microg of plasmid DNA (pDNA) or its dendrimeric or dendrosomal formulation to female Balb/c mice, followed by estimation of total IgG, IgG(1), IgG(2a), IgG(2b), biweekly. 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-gamma 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.

Stability study of stavudine-loaded O-palmitoyl-anchored carbohydrate-coated liposomes.

The purpose of this study was to evaluate the physicochemical stability of carbohydrate-anchored liposomes. In the present study, carbohydrate (galactose, fucose, and mannose) was palmitoylated and anchored on the surface of positively charged liposomes (PL). The stabilities of plain neutral liposomes (NL), PL, and O-palmitoyl carbohydrate-anchored liposomes were determined. The effects of storage conditions (4°C±2°C, 25°C±2°C/60%±5% relative humidity [RH], or 40°C±2°C/75%±5% RH for a period of 10, 20, and 30 days) were observed on the vesicle size, shape, zeta potential, drug content, and in vitro ligand agglutination assay by keeping the liposomal formulations in sealed ambercolored vials (10-mL capacity) after flushing with nitrogen. The stability of liposomal formulations was found to be temperature dependent. All the liposomal formulations were found to be stable at 4°C±2°C up to 1 month. Storage at 25°C±2°C/60%±5% RH and 40°C±2°C/75%±5% RH adversely affected uncoated liposomal formulations. Carbohydrate coating of the liposomes could enhance the stability of liposomes at 25°C±2°C/60%±5% RH and 40°C±2°C/75%±5% RH.

Radiolabeling, pharmacoscintigraphic evaluation and antiretroviral efficacy of stavudine loaded 99mTc labeled galactosylated liposomes

The study was aimed to optimize radiolabeling with 99mTc, to determine the antiretroviral activity and to study the biodistribution of 99mTc labeled galactosylated liposomes loaded with stavudine. Liposomes were prepared using reverse-phase evaporation method followed by extrusion through 200nm polycarbonate membranes. The galactosylated liposomes were assessed for in vitro ligand-specific activity and the aggregation of galactosylated liposomes was found to increase as lectin concentration was increased from 5microg/ml to 30microg/ml. Free stavudine and stavudine loaded plain and galactosylated liposomes were radiolabeled with 99mTc by direct labeling method using stannous chloride as a reducing agent. Labeling method was optimized for stannous chloride quantity to achieve maximum labeling efficiency >95%. Antiretroviral activity was determined using human immunodeficiency virus-1 (HIV) infected MT2 cell line. A dose-dependent inhibition of p24 production was observed upon treatment of HIV-1 infected MT2 cells with stavudine loaded liposomes and galactosylated liposomes. Scintigraphic imaging and quantitative biodistribution of 99mTc labeled drug and liposomes showed that liposomal formulations were better taken up by the liver and spleen. Free drug solution was cleared from the blood. Further, a significantly higher (P<0.05) liver and spleen retention was observed over a period of 24h in case of galactosylated liposomes as compared to free drug and plain liposomes. Reduced uptake of the galactosylated liposomes in bone and higher and prolonged accumulation in mononuclear phagocyte system (MPS)-rich organs indicates the excellent potential of this formulation in the treatment of HIV infection.

Ethinylestradiol-loaded ultraflexible liposomes: pharmacokinetics and pharmacodynamics.

This study aimed to develop ultraflexible liposomes as an alternative to the oral route, which would enhance the bioavailability and reduce the toxicity of ethinylestradiol. Ultraflexible liposomes of ethinylestradiol using an optimized concentration of surfactants were prepared and characterized in vitro. The effect of surfactant type under non‐occlusive conditions on transdermal permeability was assessed. A histopathological study was performed to assess the action of ethinylestradiol on the uterus and ovaries. The pharmacokinetics of free ethinylestradiol (following single oral administration and one day of application to the skin), ultraflexible liposomal ethinylestradiol and non‐flexible liposomal ethinylestradiol were studied in female Sprague—Dawley rats. Insignificant differences in size between the ultraflexible liposomal formulations containing optimized concentrations of different surfactants were observed. Ultraflexible liposomes can penetrate through pores much smaller than their own diameter. The transdermal permeability of lipophilic surfactant was greater than that of hydrophilic surfactant. The release of ethinylestradiol from the proposed formulation through rat skin was found to be constant. The histopathological study showed that the ultraflexible liposomal transdermal drug delivery system for ethinylestradiol provided effective contraception by follicular cell lysis, depletion of zona granulosa and ova, and by increasing the uterine mucosal and endometrial proliferation. Encapsulation of ethinylestradiol in ultraflexible liposomes modified the pharmacodynamics and pharmacokinetics of the contraceptive agent, resulting in a marked improvement in bioavailability and optimized therapy.

Toxicological investigation of surface engineered fifth generation poly (propyleneimine) dendrimers in vivo

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: 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.