Showing papers in "Journal of Drug Targeting in 2005"

Polysorbate-stabilized solid lipid nanoparticles as colloidal carriers for intravenous targeting of drugs to the brain: Comparison of plasma protein adsorption patterns

Abstract: Plasma proteins enriched on the surface of drug-delivery-purpose nanoparticles are regarded as key factors for determination of in vivo organ distribution after intravenous injection. Polysorbate 80-coated polybutylcyanoacrylate (PBCA) nanoparticles, preferentially adsorbing apolipoprotein E (apoE) on their surface, have previously been considered to deliver various drugs to the brain. In the present study, in vivo well tolerable solid lipid nanoparticles (SLN) using different types of polysorbates as stabilizers were produced. The influence of the different surfactants on in vitro adsorption of human plasma proteins was investigated using two-dimensional polyacrylamide gel electrophoresis (2-DE). Possible correlations of different amounts of adsorbed apoE to the hydrophilic-lipophilic balance (HLB) of the polysorbates are shown and discussed. Apolipoprotein C-II, albumin and immunoglobulin G, which are also decisive plasma proteins with regard to site-specific drug delivery of intravenously injected carriers to the brain, are compared with regard to adsorption. Moreover, certain similarities to the plasma protein adsorption patterns of previously analysed brain-specific PBCA nanoparticles could be detected. Despite some differences in adsorption behavior of proteins on the surface of polysorbate-stabilized SLN and PBCA nanoparticles, we conclude that in both cases polysorbate 80 might have the highest potential to deliver drugs to the brain.

Polymeric micelles for delivery of poorly soluble drugs: preparation and anticancer activity in vitro of paclitaxel incorporated into mixed micelles based on poly(ethylene glycol)-lipid conjugate and positively charged lipids.

Abstract: Paclitaxel-loaded mixed polymeric micelles consisting of poly(ethylene glycol)-distearoyl phosphoethanolamine conjugates (PEG-PE), solid triglycerides (ST), and cationic Lipofectin® lipids (LL) have been prepared. Micelles with the optimized composition (PEG-PE/ST/LL/paclitaxel=12/12/2/1 by weight) had an average micelle size of about 100 nm, and zeta-potential of about −6 mV. Micelles were stable and did not release paclitaxel when stored at 4°C in the darkness (just 2.9% of paclitaxel have been lost after 4 months with the particle size remaining unchanged). The release of paclitaxel from such micelles at room temperature was also insignificant. However, at 37°C, approx. 16% of paclitaxel was released from PEG-PE/ST/LL/paclitaxel micelles in 72 h, probably, because of phase transition in the ST-containing micelle core. In vitro anticancer effects of PEG-PE/ST/LL/paclitaxel and control micelles were evaluated using human mammary adenocarcinoma (BT-20) and human ovarian carcinoma (A2780) cell lines. Pacli...

Polypropylenimine dendrimer-induced gene expression changes: the effect of complexation with DNA, dendrimer generation and cell type.

Abstract: Polypropylenimine (PPI) dendrimers appear attractive non-viral vectors for the delivery of genes, antisense oligonucleotides, and small interfering RNA (siRNA). However, the effects of these synthe...

Doxorubicin loaded pH-sensitive micelle targeting acidic extracellular pH of human ovarian A2780 tumor in mice.

Abstract: The purpose of this study was to examine the efficacy of a chemotherapeutic drug, doxorubicin (DOX), loaded in pH-sensitive micelles poly(l-histidine) (M(n):5K)-b-PEG (M(n):5K) micelles. The micelles were designed to target the acidic extracellular pH of solid tumors. Studies of pH-dependent cytotoxicity, growth rate of the tumor, pharmacokinetics and biodistribution were conducted. In vitro DOX uptake upon A2780 cells by incubating the cells in a pH 6.8 complete medium at a concentration of 20 microg DOX/ml in the micelle formulation was more than five times that of pH 7.4 condition for initial 20 min. In vivo pharmacokinetic data showed that AUC (area under concentration curve) and half life time (t(1/2)) (plasma half life) of DOX in the pH sensitive micelles increased about 5.8- and 5.2-fold of free DOX in phosphate buffered saline (PBS), respectively. It appeared that DOX in the pH-sensitive micelles preferentially accumulated in the tumor site. The distributions at 12 h post injection in other organs including liver, kidney, spleen, lung and heart were not significantly different from those of DOX in PBS at a 6 mg DOX/kg dose. The in vivo test of anti-tumor activity was performed with human ovarian carcinoma A2780 which was subcutaneously xenografted in female nu/nu athymic mice. The pH-sensitive micelle formulation significantly retarded tumor growth rate without serious body weight loss. The triggered drug release by the reduced tumor pH is believed to be a major mechanism of the observed efficacy after passive accumulation of the micelles by EPR effect. This may have resulted in a local high dose of drug in the tested solid tumor.

Transferrin coupled liposomes as drug delivery carriers for brain targeting of 5-florouracil.

Abstract: Diseases and disorders of the brain are extremely difficult to treat pharmacologically because most drugs are unable to pass across the blood–brain barriers. Complex multi-strand tight junctions between adjacent cerebral endothelial cells and between choroid plexus epithelial cells form a physical barrier and prevent the passage of water soluble drugs from the blood into the brain, whereas the inward passage of lipid soluble drugs is restricted by drug efflux pumps which act as a functional barrier. In the present work, a transferrin-coupled liposomal system for brain delivery of 5-florouracil has been investigated.5-florouracil and 99mTc-DTPA bearing non-coupled liposomes were prepared by cast film method, which were coupled with the transferrin by incubating these liposomes with transferrin in the presence of the 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride in saline phosphate buffer (pH 7.4). These liposomal systems were characterized for vesicle size, percent drug entrapment, and in vi...

Intranasal mucoadhesive microemulsions of zolmitriptan: preliminary studies on brain-targeting.

Abstract: The aim of this investigation was to prepare microemulsions containing zolmitriptan (ZT) for rapid drug delivery to the brain to treat acute attacks of migraine and to characterize microemulsions and evaluate biodistribution in rats. Zolmitriptan microemulsions (ZME) were prepared using the titration method and were characterized for globule size distribution and zeta potential. ZT was radiolabeled using 99mTc (technetium) and radiolabeled-drug formulations of ZT were used to carry out biodistribution of drug in the brain of Swiss albino rats after intranasal and intravenous administration. The pharmacokinetic parameters, drug targeting efficiency (%DTE) and direct nose-to-brain drug transport (%DTP) were calculated. Brain scintigraphy imaging in rats were also performed to ascertain the uptake of drug into the brain. ZME were transparent and stable with mean globule size of 35 ± 25 nm and zeta potential of − 38– − 52 mV. 99mTc-labeled-drug formulations of ZT were found to be stable and suitable to perfor...

Targeting of daunomycin using biotinylated immunoliposomes: pharmacokinetics, tissue distribution and in vitro pharmacological effects.

Abstract: Biotinylated immunoliposomes were prepared by a non-covalent (biotin-streptavidin) coupling procedure and conjugated to the OX26 monoclonal antibody directed against the rat transferrin receptor. In vitro, these biotinylated immunoliposomes were used to by-pass P-glycoprotein in multidrug-resistant RBE4 brain capillary endothelial cells and thereby to achieve 2- to 3-fold higher intracellular accumulation of liposomal daunomycin as compared to free drug. The extent of cellular uptake of liposomal daunomycin was dose- and time-dependent, was inhibited by competition with unbound OX26 and was associated with a pharmacological (i.e. cytotoxic) effect. Cytotoxic effects of liposomal formulations of daunomycin, in contrast to the free drug, were apparent only after prolonged incubation periods being indicative of a slow intracellular unpacking and release of liposomal daunomycin. Pharmacokinetics and tissue distribution studies in the rat revealed brain accumulation of daunomycin in OX26-immunoliposomes to higher levels as compared to brain uptake of free daunomycin, or daunomycin incorporated within pegylated liposomes or within unspecific IgG(2a) isotype control immunoliposomes. Such OX26-mediated effects were not observed in other tissues such as spleen, liver, muscle or kidney.

Physicochemical Characteristics and In Vivo Deposition of Liposome-encapsulated Tea Catechins by Topical and Intratumor Administrations

Abstract: Tea polyphenols, including (+)-catechin, (−)-epicatechin, and (−)-epigallocatechin-3-gallate (EGCG), have been shown to possess potent antioxidant and anticancer activities. The aim of this study was to evaluate the possibility of using liposomes for the local delivery, including skin and tumor deposition, of these polyphenols. Liposomes containing egg phosphatidylcholine, cholesterol, or anionic species were prepared by a solvent evaporation method and then were subjected to a probe sonicator. The size, zeta potential and entrapment efficiency of these liposomal formulations were determined to provide correlations with results from a subsequent in vivo study. The release rate study showed that inclusion of an anionic species, such as deoxycholic acid (DA) or dicetyl phosphate (DP), increased the permeability of the lipid bilayers, leading to the rapid release of these formulations. No significant increase in skin deposition of catechins was observed after topical application of liposomes. On the other ha...

Body distribution of polysorbate-80 and doxorubicin-loaded [14C]poly(butyl cyanoacrylate) nanoparticles after i.v. administration in rats.

Abstract: Previously it was shown that poly(butyl cyanoacrylate) (PBCA) nanoparticles coated with polysorbate 80 are able to cross the blood-brain barrier (BBB) after i.v. administration. The objective of the present study was to investigate the influence of polysorbate 80 and doxorubicin-loading on the body distribution in rats. The biodistribution profile and brain concentration of 14C-radiolabeled PBCA nanoparticles, polysorbate 80 coated 14C-PBCA nanoparticles, and doxorubicin-loaded 14C-PBCA nanoparticles were determined by radioactivity counting after i.v. administration in rats. The 14C-PBCA nanoparticles showed a significant accumulation in the organs of the reticuloendothelial system (RES). Polysorbate 80 coating of the 14C-PBCA nanoparticles decreased this accumulation to about 40% after 1 h post injection. The brain concentration was increased about 2-fold after polysorbate 80-coating at this time point. The presence of doxorubicin in this preparation, however, decreased the brain concentration to levels...

Cell-surface display of E7 antigen from human papillomavirus type-16 in Lactococcus lactis and in Lactobacillus plantarum using a new cell-wall anchor from lactobacilli.

Abstract: The human papillomavirus type-16 (HPV-16) E7 protein is considered a major viral oncoprotein involved in cervical cancer (CxCa) and a potential candidate for the development of a vaccine against this neoplasia. Here, two lactic acid bacteria (the model one Lactococcus lactis and a probiotic one Lactobacillus plantarum) were engineered to deliver an E7 mutant protein (E7mm), which has a reduced transforming activity and consequently, could fit better to therapeutic use in humans than the native form of E7. An efficient cell-surface display of E7mm was obtained in L. lactis using an expression cassette encoding a precursor composed of (i) the signal peptide and the first 15 amino acids of the mature part of the lactococcal Usp45 protein; (ii) E7mm and (iii) the cell-wall anchor of the Streptococcus pyogenes M6 protein (CWA(M6)). This hybrid precursor was produced but not cell-wall anchored in Lb. plantarum. We thus replaced CWA(M6) by the cell-wall anchor of a Lb. plantarum protein which allows an efficient cell-wall anchoring of E7mm in this bacterium. The E7mm production and cell-surface display in both L. lactis and a probiotic bacterium, Lb. plantarum, represent one more step towards the development of a safe and effective treatment against CxCa.

Cutaneous DNA delivery and gene expression in ex vivo human skin explants via wet-etch microfabricated microneedles

Abstract: Microneedle arrays increase skin permeability by forming channels through the outer physical barrier, without stimulating pain receptors populating the underlying dermis. It was postulated that microneedle arrays could facilitate transfer of DNA to human skin epidermis for cutaneous gene therapy applications. Platinum-coated “wet-etchâ€� silicon microneedles were shown to be of appropriate dimensions to create microconduits, approximately 50 I¼m in diameter, extending through the stratum corneum (SC) and viable epidermis. Following optimisation of skin explant culturing techniques and confirmation of tissue viability, the ability of the microneedles to mediate gene expression was demonstrated using the I²-galactosidase reporter gene. Preliminary studies confirmed localised delivery, cellular internalisation and subsequent gene expression of pDNA following microneedle disruption of skin. A combination of this innovative gene delivery platform and the ex vivo skin culture model will be further exploited to optimise cutaneous DNA delivery and address fundamental questions regarding gene expression in skin.

Efficient drug delivery to atherosclerotic lesions and the antiatherosclerotic effect by dexamethasone incorporated into liposomes in atherogenic mice

Abstract: In order to confirm the efficacy of dexamethasone (DXM) incorporated into liposomes (DXM-liposomes) on atherosclerosis, drug delivery to atherosclerotic lesions and the antiatherosclerotic effect by DXM-liposomes were investigated in atherogenic mice. DXM-liposomes were prepared with egg yolk phosphatidylcholine, cholesterol and dicetylphosphate in a lipid molar ratio of 7/2/1 by the hydration method and then adjusted to three different particle sizes to clarify the influence of particle size on the drug delivery to atherosclerotic lesions and the effect on atherosclerosis. The particle sizes of DXM-liposomes were 519 nm (L500), 202 nm (L200) and 68.6 nm (L70), respectively. In both size, DXM concentration and DXM/lipid molar ratio in DXM-liposomes suspension were 1 mg DXM/ml and 0.134 mol DXM/mol total lipids, respectively. Atherogenic mice used as an experimental model develop an atherosclerotic lesion in the aorta and they were prepared by feeding an atherogenic diet for 14 weeks. The aortic pharmacokinetics of DXM-liposomes was examined by intravenous administration to atherogenic mice. The aortic uptake clearance of DXM in atherogenic mice treated with L200 was 2.6--3.2 fold greater than that in animals treated with L500, L70 or free DXM (f-DXM). Furthermore, the effects of DXM-liposomes on atherosclerosis were examined by intravenous administration to atherogenic mice once a week from 8 to 14 weeks. The antiatherosclerotic effects of DXM-liposomes were evaluated by determination of the aortic cholesterol ester (CE) level. The aortic CE level in atherogenic mice treated with L200 (55 microg DXM/kg) was significantly lower than that in animals treated with PBS. The antiatherosclerotic effect of L200 (55 microg DXM/kg) was significantly more potent than that of f-DXM (550 microg DXM/kg). These findings suggest that efficient delivery of DXM to the atherosclerotic lesions by L200 induces an excellent antiatherosclerotic effect at a lower dose. Therefore, L200 may be useful in the development of drug delivery systems for atherosclerotic therapy.

Targeted delivery of arjunglucoside I using surface hydrophilic and hydrophobic nanocarriers to combat experimental leishmaniasis

Abstract: The purpose of the present study was to investigate the therapeutic efficacy of the indigenous drug arjunglucoside I (AG) against in vivo models of experimental leishmaniasis by incorporating it in surface hydrophilic co-polymeric nanogel particles of size less than 100 nm diameter and to compare its efficacy with that of the free drug as well as the drug encapsulated in hydrophobic poly-dl-lactide (PLA) nanoparticles. The drug AG, having glucose at the terminal end of the glycosidic chain, was isolated from an indigenous source. Drug-incorporated ultra-low-sized nanogels (∼90 nm in diameter) composed of cross-linked random co-polymer of N-isopropylacrylamide (NIPAAM) and N-vinyl pyrrolidone(VP) were prepared, characterized and used as delivery vehicles to combat experimental leishmaniasis in hamster models. For comparison, drug-encapsulated hydrophobic nanoparticles (∼250 nm in diameter) made from PLA were used as a control. The drug AG was incorporated in these nanocarriers and these drug-nanocarrier co...

Antimicrobial activity of clove oil and its potential in the treatment of vaginal candidiasis.

Abstract: In the present study, we evaluated antimicrobial activity of clove oil against a range of fungal pathogens including that responsible for urogenital infection. Clove oil was found to possess strong antifungal activity against opportunistic fungal pathogens such as Candida albicans, Cryptococcus neoformans and Aspergillus fumigatus, etc. The oil was found to be extremely successful in the treatment of experimental murine vaginitis in model animals. On evaluating various formulations, topical administration of the liposomized clove oil was found to be most effective against treatment of vaginal candidiasis.

Characterization of nebulized buparvaquone nanosuspensions—effect of nebulization technology

Abstract: The poorly soluble drug buparvaquone is proposed as an alternative treatment of Pneumocystis carinii pneumonia (PCP) lung infections. Physically stable nanosuspensions were formulated in order to deliver the drug at the site of infection using nebulization. The aerosolization characteristics of two buparvaquone nanosuspensions were determined with commercial jet and ultrasonic nebulizer devices. Aerosol droplet size distribution was determined with laser diffractometry (LD). Nebulization of the nanosuspensions and dispersion media surfactant solutions produced aerosol droplets diameters in the range from 3 to 5 microm for Respi-jet Kendall, Pari Turbo Boy system and Multisonic nebulizers and particles around 9-10 microm with Omron U1. Fractions of the nanosuspensions from the nebulizer reservoir and of aerosol produced were collected to investigate changes in the size of the drug nanocrystals influenced by the nebulization technology. Comparisons were performed measuring the drug nanocrystals with photon correlation spectroscopy (PCS) and LD of the samples. Drug particle aggregates were detected in the fractions of aerosol collected from jet nebulizers. Nebulizer technology (jet vs. ultrasonic) showed influence on the stability of the drug particle size distribution of buparvaquone nanocrystals during the nebulization time evaluated.

Enhanced gene transfer activity of peptide-targeted gene-delivery vectors

Abstract: We have evaluated the capacity of the cell-binding heptapeptide SIGYPLP to enhance transgene expression using non-viral and viral gene delivery vectors. Targeted polyplex based vectors showed good levels of DNA uptake in freshly isolated human umbilical vein endothelial cells (HUVECs) compared to untargeted controls, whilst displaying only modest increases in reporter gene activity. The targeted polyplexes showed reduced levels of DNA uptake in cells of a none endothelial origin although they mediated higher levels of transgene expression. The enhanced efficiency of transgene expression may relate to the more rapid rate of cell division. However, since in vivo application of polyplexes is compromised by instability to serum proteins, serum-resistant polyplexes (surface modified with multivalent reactive hydrophilic polymers based on poly[N-(2-hydroxypropyl)methacrylamide] (pHPMA)) were also evaluated for their ability to mediate transgene expression. Surface modification of polyplexes with pHPMA ablates non-specific cell entry, reducing levels of transgene expression, whilst the incorporation of the SIGYPLP peptide into the hydrophilic polymer resulted in restored transgene expression in all formulations tested. The technology of surface modification using pHPMA can also be applied in the context of viruses, masking receptor-binding epitopes and enabling the linkage of novel cell targeting ligands, enabling construction of a virus with receptor-specific infectivity. Retargeting of adenovirus based vectors using the same polymer-peptide construct enhanced levels of transgene expression in HUVECs to greater than 15 times that observed using parental (unmodified) virus, whilst restoring levels of transgene expression in non-endothelial cell lines tested. The use of constructs based on conjugates between hydrophilic polymers and small receptor-binding oligopeptides as agents for retargeting viral or non-viral vectors to cellular receptors represents a simple alternative to the use of antibodies as targeting ligands for cell specific gene delivery.

Phage display selection for cell-specific ligands: Development of a screening procedure suitable for small tumor specimens

Abstract: Phage display technology has been widely used for developing tumor-targeting agents. Most of the efforts were directed towards identifying phage-displayed ligands against cancer-relevant purified targets and cancer cell lines. Whole cell screening procedures typically use a relatively large sample size and are not ideally suited for complex tumor tissues. We describe here a screening protocol that is suitable for non-adherent tumor cells from biopsy specimens. It requires only approximately 20,000 cells/round for biopanning and approximately 10,000 cells/well for subsequent clone binding assessment by ELISA. We standardized the newly developed protocol using erbB2-overexpressing SKBR3 breast cancer cells and compared the results with conventional protocols employing about 10-times more plate-adhered fixed or live cells. The selection rate of SKBR3-binding clones from biopanning approximately 20,000 non-adherent SKBR3 cells by our filter cup protocol was comparable to that obtained from using approximately 200,000 plate-adhered cells. Assessment of clones selected from different phage libraries showed that clones from fixed or live cells, adherent or non-adherent cells, either biopanned in filter cup or plate share specific motifs and binding properties. Some of the clones from each biopanning protocol bound to purified erbB2 and shared motifs with erbB family of receptors and their known ligands. These results demonstrated that the protocol developed in this study was capable of selecting cell-specific ligands using relatively small numbers of cells. Screening cells from a fresh human breast cancer specimen using our protocol showed enrichment of tumor binding clones at successive rounds of selection and some of the selected clones were tumor-specific in comparison to normal breast cells. These protocols have direct application to screen for tumor-binding ligands with small tumor tissue specimens.

Protective immune responses to meningococcal C conjugate vaccine after intranasal immunization of mice with the LTK63 mutant plus chitosan or trimethyl chitosan chloride as novel delivery platform.

Abstract: Chitosan and its derivative N-trimethyl chitosan chloride (TMC), given as microparticles or powder suspensions, and the non-toxic mucosal adjuvant LTK63, were evaluated for intranasal immunization with the group C meningococcal conjugated vaccine (CRM-MenC). Mice immunized intranasally with CRM-MenC formulated with chitosan or TMC and the LTK63 mutant, showed high titers of serum and mucosal antibodies specific for the MenC polysaccharide. Neither significant differences were observed between microparticle formulations and powder suspensions nor when LTK63 was pre-associated to the delivery system or not. The bactericidal activity measured in serum of mice immunized intranasally with the conjugated vaccine formulated with the delivery systems and the LT mutant was superior to the activity in serum of mice immunized sub-cutaneously. Importantly, intranasal but not parenteral immunization, induced bactericidal antibodies at the nasal level, when formulated with both delivery system and adjuvant.

Oral delivery of low-molecular-weight heparin using sodium caprate as absorption enhancer reaches therapeutic levels

Abstract: The primary objective of this study was to evaluate sodium caprate as an oral penetration enhancer for low molecular weight heparin (LMWH), ardeparin. In vitro studies using Caco-2 cell monolayer indicated that 0.0625% of sodium caprate gave approximately 2-fold enhancement of ardeparin compared to negative control with almost 100% cell survival as evaluated by MTT cytotoxicity assay. In vivo studies in rats with ardeparin (1,200 IU/kg) and sodium caprate (100 mg/kg) led to a relative bioavailability of 27% with plasma anti-factor Xa levels within the therapeutic range (>0.2 IU/ml). Moreover, under these conditions, histological examination provided evidence that there was no damage to the gastrointestinal wall. Regional permeability studies using rat intestine indicated the colon as the region of maximum permeation. These results suggest that, at the dose administered, sodium caprate acts as a relatively safe and efficient absorption enhancer in the quest for alternatives for the oral delivery of LMWH.

Water-soluble polymers for targeted drug delivery to human squamous carcinoma of head and neck

Abstract: Human squamous cell carcinoma of the head and neck (SCCHN) is characterized by over expression of a tumor cell surface-specific receptor namely Hsp47/CBP2 that makes it a favorable candidate for targeted delivery of anticancer drugs. Several synthetic peptides have been identified as effective ligands for binding to CBP2. The purpose of this study is to investigate the potential of water-soluble N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer-doxorubicin (Dox) conjugates containing a Hsp47/CBP2 binding peptide sequence, namely WHYPWFQNWAMA for targeted delivery to SCCHN. An HPMA copolymer containing Dox and CBP2 targeting peptide conjugated via lysosomally degradable glycylphenylalanylleucylglycine (GFLG) spacer was synthesized by free radical precipitation copolymerization. A control polymer without targeting moiety was also synthesized. The conjugates were characterized for drug content, peptide content, molecular weight and molecular weight distribution. The uptake of polymeric conjugates by both drug resistant and drug sensitive SCCHN cells were determined in vitro by flow cytometry using FACS scan analysis. Cytotoxicity of the conjugates towards drug sensitive as well as multidrug resistant SCCHN cells were evaluated by a clonal survival assay and compared to free Dox. The cytotoxicity of the free peptide was similarly evaluated. The internalization and subcellular fate of the conjugates in drug sensitive SCCHN cells was monitored using confocal microscopy. The new targetable copolymer contained 0.16 mmole peptide/g polymer. Studies on drug sensitive SCCHN cells demonstrated lesser uptake of both targeted and non-targeted conjugates compared to free Dox suggesting a slower endocytic mechanism of uptake for the conjugates as opposed to rapid diffusion of free Dox. At higher Dox equivalent concentrations (>20 microM) the targeted conjugate showed significantly higher uptake (p < or = 0.028) than the non-targeted conjugate. The uptake of the targeted conjugate was inhibited in the presence of an anti Hsp47 antibody suggesting the involvement of active receptor mediated endocytosis in cell entry of the conjugate. Compared to free Dox, the targeted and non-targeted conjugates caused marginally lower inhibition (p < or = 0.01) of the drug sensitive SCCHN cells. In contrast, the same conjugates showed significantly higher uptake (p < or = 0.004) by drug resistant SCCHN cells and caused significantly higher inhibition (p < or = 0.02) of drug resistant SCCHN cells when compared to free Dox. Results suggest that the polymeric conjugates were able to overcome drug resistance. Confocal microscopy studies demonstrated the uptake of the polymeric conjugates, followed by internalization, intralysosomal localization and subsequent release of Dox. HPMA copolymer-Dox-peptide conjugates targeted to SCCHN cells were able to overcome drug resistance and increase efficacy in vitro. The results suggest that targetable polymeric conjugates have potential to improve systemic head and neck cancer chemotherapy by increasing tumor localization and reducing dose-limiting toxicity.

In vitro and in vivo evaluation of pectin beads for the colon delivery of β-lactamases

Abstract: The aim of the present study was to provide a “proof of concept” of colon delivery of β-lactamases by pectin beads aiming to degrade residual β-lactam antibiotics, in order to prevent the emergence of resistant bacterial strains.Pectin beads were prepared according to ionotropic gelation method using CaCl2 as a gelling agent. Particles were then washed and soaked in polyethylenimine (PEI). Coating beads with PEI considerably improved their stability in simulated intestinal medium. In vitro studies showed that β-lactamases were released from pectin beads in colonic medium due to the action of pectinolytic enzymes. When ampicillin was added to this medium, the release of β-lactamases induced, as expected, the antibiotic inactivation. Finally, after oral administration of loaded-beads to CD1 mice, β-lactamases were retrieved in high concentrations in faeces. Observation by SEM of beads extracted from mice intestinal tracts concluded the core degradation of beads without any modification of the PEI coating la...

Targeted gene delivery to hepatoma cells using galactosylated liposome-polycation-DNA complexes (LPD)

Abstract: A major goal for gene therapy is to obtain targeted vectors that transfer genes efficiently to specific cell types. The liver possesses a variety of characteristics that make this organ very attractive for gene therapy. In the present study, four cholesterylated thiogalactosides 1a approximately d with different spacer length were synthesized to formulate novel lipid-polycation-DNA (LPD) complexes, which were composed of galactosylated cationic liposomes, protamine sulfate and plasmid DNA. The galactosylated LPD1c significantly improved the levels of gene expression in cultured hepatoma cells HepG2 and SMMC-7721, while LPD1a and LPD1b did not significantly improve the levels compared with non-galactosylated LPD. Meanwhile, increased transfection activity was not observed in mouse fibroblasts L929 for galactosylated LPDs. Cytotoxicity of galactosylated LPDs assay showed they had no obvious toxicities to L929 cells and HepG2 cells. In summary, the length of the spacer between the anchor and galactose residues was important for the recognition of asialoglycoprotein receptor. The LPD1c described here, combining the condensing effect of protamine and the targeting capability of cholesterylated thiogalactosides, are potentially useful gene carriers to liver parenchymal cells.

HPMA copolymer-aminoglutethimide conjugates inhibit aromatase in MCF-7 cell lines.

Abstract: N-(2-Hydroxypropyl)methacrylamide (HPMA) copolymer–doxorubicin (Dox) has already shown clinical activity in breast cancer patients. Moreover, we have recently found that an HPMA conjugate containing a combination of both Dox and the aromatase inhibitor aminoglutethimide (AGM) shows significantly increased anti-tumour activity in vitro. To better understand the mechanism of action of HPMA copolymer–AGM conjugates several models were used here to investigate their effect on cell growth and aromatase inhibition. Cytotoxicity of HPMA copolymer conjugates containing AGM, Dox and also the combination AGM–Dox was determined by MTT assay in MCF-7 and MCF-7ca cells. Androstenedione (5 × 10− 8 M) stimulates the growth of MCF-7ca cells. Both free AGM and polymer-bound AGM (0.2–0.4 mg/ml) were shown to block this mitogenic activity. When MCF-7ca cells were incubated [3H]androstenedione both AGM and HPMA copolymer–GFLG–AGM (0.2 mg/ml AGM-equiv.) showed the ability to inhibit aromatase. Although, free AGM was able to i...

Targeting of mannosylated liposome incorporated benzyl derivative of Penicillium nigricans derived compound MT81 to reticuloendothelial systems for the treatment of visceral leishmaniasis.

Abstract: The antileishmanial property of a Benzyl derivative of a new antibiotic MT81 (Bz2MT81), isolated and purified from a fungal strain of Penicillium nigricans NRRL 917 was tested in free, liposome intercalated and mannose coated liposome intercalated forms in vivo against visceral leishmaniasis in hamsters. Mannose grafted liposome intercalated Bz2MT81 eliminated intracellular amastigotes of Leishmania donovani within splenic macrophages more efficiently than the liposome intercalated Bz2MT81 or free Bz2MT81. At a dose equivalent to 7.5 microg/Kg body weight when injected subcutaneously (s.c) in mannose grafted liposome intercalated form for 15 days in an interval of three days, the splenic parasitic load decreased to the extent of 79.1% of the total parasite present in infected control animals. Whereas, an identical amount (7.5 mug/Kg body weight) of Bz2MT81 in free or liposome intercalated form was found less effective in controlling the parasite in spleen (in free Bz2MT81 form, suppression of parasitic load is 49.8% and in liposome intercalated form, it is 55.1%). Both mannosylated liposomes and Bz2MT81 were noted non-toxic to the host peritoneal macrophages. Histological examinations of spleen and liver, kidney function tests (SGPT, alkaline phosphatase, creatinine and urea in blood plasma) showed that the toxicity of Bz2MT81 was reduced up to normal level when mannose grafted liposomal Bz2MT81 were administered.

Sugar coated liposomal flavonoid: a unique formulation in combating carbontetrachloride induced hepatic oxidative damage.

Abstract: Rats were administered a single dose of plant origin phenolic antioxidant Quercetin (QC) in free, liposome encapsulated and galactosylated liposome encapsulated forms 2 h prior to hepatotoxic dose of carbontetrachloride (CCl4, 40% v/v in olive oil, 1 ml/kg b.wt). Among those three different forms of QC tested, only galactosylated liposomal QC provided significant protection against CCl4 induced hepatic oxidative damage. After 24 h of injection (S.C.) hepatic cells of rats were found susceptible to CCl4 induced oxidative damage and it was monitored by the increased amount of conjugated diene in hepatic membrane. The two-fold increase in conjugated diene by the induction of CCl4 was decreased upto normal level by galactosylated liposomal QC pre-treatment. Carbontetrachloride induced membrane damage in hepatic cells and it was judged by the blood serum pathological and liver tissue histopathological examination. Membrane damage by the induction of CCl4 was further evaluated by the decreased level of plasma membrane (PM) bound enzyme Na+/K+ ATPase activity and it was increased only by the pre-treatment of galactosylated liposomal QC. Carbontetrachloride induced a substantial decrease both in enzymatic and molecular endogenous antioxidant levels in hepatic cells.The depression in antioxidant system in hepatic cells was completely prevented by a single dose of galactosylated lipsosomal QC prior to CCl4 treatment. Liver uptake of QC was estimated after 2 h of the flavonoid injection (8.9 micromol/kg body weight) (free or liposomal forms) and 85% of the injected QC was found in liver in the case of galactosylated liposomal QC. Whereas only 25% of the injected dose was detected in liver when an identical amount of free QC was injected. Carbontetrachloride also induced an alteration in membrane fluidity and it was evaluated by a decrease in membrane micro-viscosity. Free QC pre-treatment resulted in no protection against CCl4 induced increase in hepatic membrane fluidity, whereas galactosylated liposomal QC exerted a significant protection against the increase. Results of this study revealed that QC in galactosylated liposome could exert a significant protection against CCl4 induced hepatocellular injury.

Preparation and characterization of a biodegradable drug targeting system for anticancer drug delivery: microsphere-antibody conjugate.

Abstract: Targeted delivery of anticancer drugs is one of the most actively pursued goals in anticancer chemotherapy. A major disadvantage of anticancer drugs is their lack of selectivity for tumour tissue, which causes severe side effects and results in low cure rates. Any strategy by which a cytotoxic drug is targeted to the tumour, thus increasing the therapeutic index of the drug, is a way of improving cancer chemotherapy and minimizing systematic toxicity. This study covers the preparation of the gelatin microsphere (GM)-anti-bovine serum albumin (anti-BSA) conjugate for the development of a drug targeting approach for anticancer drug delivery. Microspheres of 5% (w/v) gelatin content were prepared by crosslinking with glutaraldehyde (GTA) at 0.05 and 0.50% (v/v) concentration. Microspheres were in the size range of 71-141?microm. The suitability of these microspheres as drug carriers for anticancer drug delivery was investigated in vitro by studying the release profiles of loaded methotrexate (MTX) and 5-fluorouracil (5-FU) and the cytotoxicities on cancer cell lines. The in vitro MTX release profiles (approximately 22-46% released in 24 h depending on the amount of GTA used) were much slower compared to 5-FU (approximately 42-91% released in 24 h). Both drugs demonstrated an initial fast release, which was followed by gradual, sustained drug release. The MTT cytotoxicity test results of GMs loaded with 5-FU and MTX showed approximately 54-70% and approximately 52-67% cytotoxicities in 4 days. In general, incorporation of MTX and 5-FU in microspheres enhanced the cytotoxic effect in a more prolonged manner compared to the free drugs. Gelatin micospheres were chemically conjugated to anti-BSA and the antigen-antibody activities were studied by immunofluorescence. Results indicated approximately 80% binding with conjugated anti-BSA and BSA-FITC. Based on their low cytotoxicity and the high antigen binding efficiencies, anti-BSA conjugated gelatin microspheres could be suitable targeted drug carrier systems for selective and long-term delivery of anticancer drugs to a specific body compartment (i.e. bladder cancer).

Macrophage depletion induced by clodronate-loaded erythrocytes.

Abstract: Given the important role of macrophages in various disorders, the transient and organ specific suppression of their functions may benefit some patients. Until now, liposome-encapsulated bisphosphonate clodronate has been extensively proposed to this end. In this paper, we demonstrate that erythrocytes loaded with clodronate can also be effective in macrophage depletion. Here, clodronate was encapsulated in erythrocytes through hypotonic dialysis, isotonic resealing and reannealing to final concentrations of 4.1 +/- 0.4 and 10.1 +/- 0.8 micromol/ml of human and murine erythrocytes, respectively. The ability of clodronate-loaded erythrocytes to deplete macrophages was evaluated both in vitro and in vivo. In vitro studies on human macrophages showed that a single administration of engineered erythrocytes was able to reduce cell adherence capacity in a time-dependent manner, reaching 50 +/- 4% reduction, 13 days post treatment. The administration of loaded erythrocytes to cultures of murine peritoneal macrophages was able to reduce macrophage adhesion 67 +/- 3%, 48 h post treatment. In vivo, the ability of clodronate-loaded erythrocytes to deplete macrophages was evaluated both in Swiss and C57BL/6 mice. Swiss mice received 125 microg of clodronate through erythrocytes and 6 days post treatment 69 +/- 7% reduction in the number of adherent peritoneal macrophages and 75 +/- 5% reduction in number of spleen macrophages were observed. C57BL/6 mice received 220 microg clodronate by RBC and 3 and 8 days post treatment 65 +/- 7% reduction in the number of spleen macrophages and the complete depletion of liver macrophages were obtained. In summary, our results indicate that clodronate selectively targeted to the phagocytic cells by a single administration of engineered erythrocytes is able to deplete macrophages, even if not completely. The transient suppression of macrophage functions through clodronate-loaded erythrocytes can be used in many biomedical phenomena and research applications.

Rectal and vaginal administration of insulin–chitosan formulations: An experimental study in rabbits

Abstract: Insulin is a polypeptide drug and it is degraded by gastrointestinal enzymes, therefore, it cannot be used via oral route readily. There are only parenteral forms available in the market. The aim of this study was to investigate the effect of rectal and vaginal administration of various insulin gel formulations on the blood glucose level as alternative routes in rabbits. Chitosan gel (CH-gel) was used as a carrier; the penetration enhancing effect of sodium taurocholate and dimethyl-beta-cyclodextrin (DM-betaCD) was also investigated. CH-gel provided longer insulin release. The maximum decreasing effect on blood glucose level was observed with insulin-CH-gel containing 5% DM-betaCD. In conclusion, our results indicate that insulin may penetrate well through the rectal and vaginal mucosae from the CH-gel. DM-betaCD was also found to be a useful agent to enhance the penetration of insulin through rectal and vaginal membranes.

Generation of immunoliposomes using recombinant single-chain Fv fragments bound to Ni-NTA-liposomes.

Abstract: Recombinant single-chain Fv antibody fragments (scFv) can be combined with liposomes to generate immunoliposomes for targeted drug delivery. Recent studies have shown that scFv molecules modified to express a C-terminal cysteine residue can be used for site-directed chemical conjugation. Here, we present a new method by immobilizing scFv fragments via their C-terminal hexahistidyl-tag on liposomes containing Ni-NTA-lipids (Ni-NTA-DOGS) in their lipid bilayer without the need to introduce additional reactive groups in the protein. Using an anti-endoglin scFv as a model antibody, we could show that scFv molecules are efficiently immobilized on the liposome surface and that these immunoliposomes bind specifically and strongly to endoglin-expressing endothelial cells. This approach allows for a rapid and flexible generation of target cell-specific immunoliposomes.

Biodistribution characteristics of mannosylated and fucosylated O/W emulsions in mice.

Abstract: Cell-specific drug delivery is one of the most promising strategies for improving therapeutic efficiency and minimizing systemic toxicity. Carrier systems devoted to receptor-mediated targeting need to be developed. In the case of liver-non-parenchymal cell-specific targeting systems, glycosylated emulsions have been developed as carriers for lipophilic drugs and/or peptides. This present study demonstrates the in vivo disposition behaviour and pharmacokinetic characteristics of mannosylated (Man-) and fucosylated (Fuc-) emulsions incorporated with cholesten-5-yloxy-N-(4-((1-imino-2-D-thiomannosylethyl)amino)alkyl)formamide (Man-C4-Chol) and its fucosylated derivatives (Fuc-C4-Chol), respectively. Man- (or Fuc-) emulsions are composed of soybean oil, EggPC and Man-C4-Chol (or Fuc-C4-Chol) in a weight ratio of 70:25:5. After intravenous administration to mice, these two types of [(3)H]cholesteryl hexadecyl ether (CHE)-labelled glycosylated emulsions were rapidly eliminated from the blood circulation and preferentially recovered in the liver. In contrast, bare (Bare-) emulsions composed of soybean oil:EggPC:cholesterol (Chol) in a weight ratio of 70:25:5 were more retained in the blood circulation. The hepatic uptake clearances of Man- and Fuc-emulsions were 3.3- and 4.0-times greater than that of Bare-emulsions. Interestingly, the hepatic uptake clearance of Fuc-emulsions was significantly higher that that of Man-emulsions. The uptake ratios by non-parenchymal cells (NPC) and parenchymal cells (PC) (NPC/PC ratio) for Bare-, Man- and Fuc-emulsions were found to be 0.4, 2.0 and 2.9, respectively. The hepatic uptakes of [(3)H]CHE-labelled Man- and Fuc-emulsions were reduced by pre-dosing with glycosylated proteins and liposomes. These results clearly support the conclusion that Man- and Fuc-emulsions are promising carrier systems for liver NPC-specific targeting via receptor-mediated mechanism.