pH-Sensitive PEGylated Liposomal Silybin: Synthesis,In Vitro and In Vivo Anti-Tumor Evaluation

The drug delivery systems improve the efficacy of chemotherapeutics through enhanced targeting and controlled release however, biological barriers of tumor microenvironment greatly impede the penetration of nanomedicine within the tumor. We report herein the fabrication of a PEG-detachable silybin (SLB) pH-sensitive liposome decorated with TAT-peptide. For this, Acyl hydrazide-activated PEG₂₀₀₀ was prepared and linked with ketone-derivatized DPPE via an acid-labile hydrazone bond to form mPEG₂₀₀₀-HZ-DPPE. TAT peptide was conjugated with a shorter -PEG₁₀₀₀-DSPE spacer and post-inserted into PEGylated liposome (DPPC: mPEG₂₀₀₀–DSPE: Chol). To prepare nanoliposomes (around 100 nm), first, a novel method was used to prepare SLB-Soya PC (SLB-SPC) complex, then this complex was incorporated into nanoliposomes. The pH-sensitivity and shielding effect of long PEG chain on TAT peptide was investigated using DiI liposome and FACS analysis. Pre-treatment to the lowered pH enhanced cellular association of TAT-modified pH-sensitive liposome due to the cleavage of hydrazone bond and TAT exposure. Besides, TAT-modified pH-sensitive liposomes significantly reduced cell viability compared to the plain liposome. In vivo results were very promising with pH-sensitive liposome by detaching PEG moieties upon exposure to the acidic tumor microenvironment, enhancing cellular uptake, retarding tumor growth, and prolonging the survival of 4T1 breast tumor-bearing BALB/c mice. TAT modification of pH-sensitive liposome improved cancer cell association and cytotoxicity and demonstrated potential intracellular delivery upon exposure to acidic pH. However, in in vivo studies, TAT as a targeting ligand significantly decreased the therapeutic efficacy of the formulation attributed to an inefficient tumor accumulation and higher release rate in the circulation. The results of this study indicated that pH-sensitive liposome containing SLB, which was prepared with a novel method with a significant SLB loading efficiency, is very effective in the treatment of 4T1 breast tumor-bearing BALB/c mice and merits further investigation.     

Mucoadhesive Microspheres for Gastroretentive Delivery of Acyclovir: In Vitro and In Vivo Evaluation

The aim of the present investigation was to evaluate the potential use of mucoadhesive microspheres for gastroretentive delivery of acyclovir. Chitosan, thiolated chitosan, Carbopol 71G and Methocel K15M were used as mucoadhesive polymers. Microsphere formulations were prepared using emulsion-chemical crosslinking technique and evaluated in vitro, ex-vivo and in-vivo. Gelatin capsules containing drug powder showed complete dissolution (90.5 +/- 3.6%) in 1 h. The release of drug was prolonged to 12 h (78.8 +/- 3.9) when incorporated into mucoadhesive microspheres. The poor bioavailability of acyclovir is attributed to short retention of its dosage form at the absorption sites (in upper gastrointestinal tract to duodenum and jejunum). The results of mucoadhesion study showed better retention of thiolated chitosan microspheres (8.0 +/- 0.8 h) in duodenal and jejunum regions of intestine. The results of qualitative and quantitative GI distribution study also showed significant higher retention of mucoadhesive microspheres in upper GI tract. Pharmacokinetic study revealed that administration of mucoadhesive microspheres could maintain measurable plasma concentration of acyclovir through 24 h, as compared to 5 h after its administration in solution form. Thiolated chitosan microsphere showed superiority over the other formulations as observed with nearly 4.0-fold higher AUC(0-24) value (1,090 +/- 51 ng h/ml) in comparison to drug solution (281 +/- 28 ng h/ml). Overall, the result indicated prolonged delivery with significant improvement in oral bioavailability of acyclovir from mucoadhesive microspheres due to enhanced retention in the upper GI tract.     

Formulation and In Vitro Characterization of Thermosensitive Liposomes for the Delivery of Irinotecan

The effect of the lipid composition on the physicochemical properties of a liposomal carrier containing irinotecan (CPT-11) and its in vitro antitumoral activity on a colon cancer cell line has been investigated. The paper describes the procurement of a novel and easy-to-prepare temperature-sensitive carrier for CPT-11 and proves its suitability as delivery system on the basis of its ability to incorporate the drug and on its efficiency to promote drug internalization. Permeability data, studied in vitro in a simulated biological medium at different temperatures, showed that both the nature of the lipids and the ratio in which they have been mixed play a key role in the release of the encapsulated product. Stable temperature-sensitive liposomes with good drug incorporation efficiency were obtained and characterized. The cellular uptake and the cytotoxic activity of the CPT-11 liposomal formulation were compared with those corresponding to the free drug with promising results, being concentration and time dependent and significantly higher. Thus, it could be interpreted that the greatest cytotoxic effect of liposomal CPT-11 was because of the increased uptake provided by the liposomal carrier. Studies of cell cycle and annexin V binding showed drug-induced changes in cell cycle dynamics and the proapoptotic effect of liposomal CPT-11. © 2014 Wiley Periodicals, Inc. and the American Pharmacists Association J Pharm Sci 103:3127–3138, 2014     

Enhancement of Transdermal Iontophoretic Delivery of a Liposomal Formulation of Colchicine by Electroporation

Iontophoresis of colchicine solution through electroporated skin showed maximum enhancement as compared to iontophoresis and electroporation alone. Encapsulation of colchicine in positively charged liposomes further augmented the delivery,with the amount of drug in the receptor after 24 h being 1348 342 mug/cm2 when iontophoresis was performed through electroporated skin as compared to 666 38mug/cm2 when it was performed through nonelectroporated skin and 41 18mug/cm2 when only electroporation was performed. The impedance of the skin was observed to drop sharply due to electroporation, with a postpulse recovery of about 30% over 24h. Also the total amount transported was compared to the total charge delivered in the case of each of the protocols. Hence this serves as initial evidence for potential of charged liposomes for the enhanced transdermal delivery of nonionized or neutral drugs using a combination of electroporation and iontophoresis.     

Enhancement of Transdermal Iontophoretic Delivery of a Liposomal Formulation of Colchicine by Electroporation

Iontophoresis of colchicine solution through electroporated skin showed maximum enhancement as compared to iontophoresis and electroporation alone. Encapsulation of colchicine in positively charged liposomes further augmented the delivery,with the amount of drug in the receptor after 24 h being 1348 342 mug/cm2 when iontophoresis was performed through electroporated skin as compared to 666 38mug/cm2 when it was performed through nonelectroporated skin and 41 18mug/cm2 when only electroporation was performed. The impedance of the skin was observed to drop sharply due to electroporation, with a postpulse recovery of about 30% over 24h. Also the total amount transported was compared to the total charge delivered in the case of each of the protocols. Hence this serves as initial evidence for potential of charged liposomes for the enhanced transdermal delivery of nonionized or neutral drugs using a combination of electroporation and iontophoresis.     

In Vivo and Ex Vivo Evaluation of a Multi-Particulate Composite Construct for Sustained Transbuccal Delivery of Carbamazepine

Carbamazepine (CBZ) is a leading molecule in the management of epilepsy. Surveys have revealed that a sufficient lack of therapeutically efficient CBZ transbuccal formulation exists. Therefore, this investigation was directed toward designing multiparticulate composite construct (MCC) for the transbuccal delivery of CBZ. The MCC was formulated using interphase, coparticulate–cosolvent homogenization technique, and lyophilization. In vitro, ex vivo, and in vivo investigations were performed. The mesoporous (pore width = 80.1233 Å) MCC was mechanically stable (Є_D = 0.0290 J, M_F = 8.5490 N/mm) and resilient (M_R = 5.5040%). It demonstrated distinctive controlled release (9.9800%/h), permeation enhancing (10.8730%/h), drug loading (90.0541%), and bioadhesive (ω_adh = 0.0034 J, F_det = 1.0751 N) capacities. In vivo studies on pigs showed the ability of the MCC to effectively initiate and regulate transbuccal permeation of CBZ as visualized by outcomes of the quantitative and qualitative assessments of isolated plasma samples. Furthermore, comparisons of in vitro and in vivo data of MCC with a conventional product highlighted its capability to attain higher bioavailability and more controlled release trends. Histological and cytological investigations confirmed that the MCC is biocompatible. The mathematical model produced relevant pharmacokinetics and in vitro/in vivo correlation information. © 2014 Wiley Periodicals, Inc. and the American Pharmacists Association J Pharm Sci 103:1157–1169, 2014     

In Vitro and In Vivo Evaluation of Lipofufol, a New Triple Stealth Liposomal Formulation of Modulated 5-Fu: Impact on Efficacy and Toxicity

Purpose

Drug resistance and severe toxicities are limitations when handling 5-FU. We have developed a triple liposomal formulation of 5-FU combined to 2′-deoxyinosine and folinic acid to improve its efficacy-toxicity balance.

Methods

Stealth liposomes were obtained using the thin-film method. Antiproliferative activity was tested on human colorectal and breast cancer models using sensitive (HT29) and resistant (SW620, LS174t, MDA231) cell lines. In vivo, pharmacokinetics, biodistribution and safety studies were performed in rodents. Finally, efficacy was evaluated using two tumor-bearing mice models (LS174 and MDA231) with response and survival as main endpoints.

Results

LipoFufol is a 120-nm pegylated liposome, displaying 20–30% encapsulation rates. In vitro, antiproliferative activities were higher than 5-FU, and matched that of FolFox combination in colorectal models, but not in breast. Drug monitoring showed an optimized pharmacokinetics profile with reduced clearance and prolonged half-life. Liposome accumulation in tumors was shown by fluorescence-based biodistribution studies. Beside, milder neutropenia was observed when giving LipoFufol to animals with transient partial DPD-deficiency, as compared with standard 5-FU. In LS174t-bearing mice, higher response and 55% longer survival were achieved with Lipofufol, as compared with 5-FU.

Conclusion

The issues of drug-resistance and drug-related toxicity can be both addressed using a stealth liposomal formulation of modulated 5-FU.     

In Vitro Cutaneous and Percutaneous Delivery and in Vivo Efficacy of Tetracaine from Liposomal and Conventional Vehicles

The aim of this study was to quantitate drug delivery and correlate drug concentration in the skin with anesthetic effect. The rate of delivery of radiolabelled tetracaine from two different liposome formulas (1F2 and 23C2) and two conventional dosage forms (PEG Ointment USP and Glaxal^® base) was investigated in Flow-thru diffusion cells using human breast skin from mammoplasty. The results indicated a 1.5 and 4 times higher concentration of tetracaine within the skin when the liposomal formula (1F2) was used, compared to tetracaine in Glaxal^® base and PEG Ointment USP, respectively. The amount of drug delivered into the skin in 24 h from the liposomal formula was 5.3% of total applied whereas from Glaxal^® base it was 3.3% and from PEG Ointment base it was 1.2%. The amount of liposomal (1F2) phospholipids in the skin after 24h was 68.3 µg/cm² (0.2% of total applied). The steady state flux of tetracaine from liposomes (1F2) was 16.06 µg/cm²/h with a lag time of 3.1h, from Glaxal^® base 10.24 µg/cm/h with a lag time of 11.2h and from PEG Ointment it was 5.70 µg/cm²/h with a lag time of 9.0h. The second liposome formula (23C2) showed similar flux and permeability coefficient than the Glaxal^® base, however the lag time was about half. The results indicated that optimized liposome formulation is necessary to achieve maximum drug delivery. The concentration of drug within the skin and the flux measured in vitro showed correlation with in vivo efficacy. The in vivo data showed that liposomal (1F2) tetracaine produced the deepest anesthesia with shortest onset in volunteers, followed by Glaxal^® base, liposome formula 23C2 while tetracaine in PEG Ointment had a lack of effect.     

DRV Liposomal Bupivacaine: Preparation,Characterization, and In Vivo Evaluation in Mice

Purpose. To evaluate the dehydration-rehydration technique to prepare a formulation of liposomal bupivacaine, and to assess its analgesic efficacy. Methods. Bupivacaine hydrochloride (BUP) was encapsulated into dehydration-rehydration vesicles (DRV) of varying phospholipid (PL) compositions. Two bilayer-forming phospholipids were used, the fluid dimyristoyl-phosphatidylcholine and the solid dis- tearoyl-phosphatidylcholine (DSPC), with 20 or 40 mol% cholesterol, in the presence of bupivacaine at a 1.28 or 0.64 BUP/PL mole ratio. After rehydration, drug/lipid ratios were determined. The formulation with the highest drug/lipid ratio (DSPC/cholesterol in an 8:2 mole ratio prepared in the presence of bupivacaine in a 1.28 BUP/PL mole ratio) was adjusted to a final bupivacaine concentration of 3.5% or 0.5%. The duration of skin analgesia after subcutaneous injection in mice produced by these formulations was compared with the conventional administration of a plain 0.5% solution of BUP. In addition, the concentration of residual bupivacaine at the injection site was followed for 96 h. Results. The relatively low organic solvent/aqueous phase and membrane/aqueous phase partition coefficients, together with liposomal trapped volume and BUP/PL mole ratio, indicated that most of the drug was encapsulated in the intraliposome aqueous phase of the DRV. The DSPC/cholesterol 8:2 mole ratio had the best drug encapsulation (BUP/PL = 0.36). Compared to plain BUP, these BUP-DRV produced significant prolongation of analgesia, which is explained by longer residence time of the drug at the site of injection. Conclusions. Bupivacaine-DRV may have a role in achieving safe, effective, and prolonged analgesia in humans.     

In Vitro and In Vivo Evaluation of a Water-in-Oil Microemulsion System for Enhanced Peptide Intestinal Delivery

Peptide and protein drugs have become the new generation of therapeutics, yet most of them are only available as injections, and reports on oral local intestinal delivery of peptides and proteins are quite limited. The aim of this work was to develop and evaluate a water-in-oil (w/o) microemulsion system in vitro and in vivo for local intestinal delivery of water-soluble peptides after oral administration. A fluorescent labeled peptide, 5-(and-6)-carboxytetramethylrhodamine labeled HIV transactivator protein TAT (TAMRA-TAT), was used as a model peptide. Water-in-oil microemulsions consisting of Miglyol 812, Capmul MCM, Tween 80, and water were developed and characterized in terms of appearance, viscosity, conductivity, morphology, and particle size analysis. TAMRA-TAT was loaded and its enzymatic stability was assessed in modified simulated intestinal fluid (MSIF) in vitro. In in vivo studies, TAMRA-TAT intestinal distribution was evaluated using fluorescence microscopy after TAMRA-TAT microemulsion, TAMRA-TAT solution, and placebo microemulsion were orally gavaged to mice. The half-life of TAMRA-TAT in microemulsion was enhanced nearly three-fold compared to that in the water solution when challenged by MSIF. The treatment with TAMRA-TAT microemulsion after oral administration resulted in greater fluorescence intensity in all intestine sections (duodenum, jejunum, ileum, and colon) compared to TAMRA-TAT solution or placebo microemulsion. The in vitro and in vivo studies together suggested TAMRA-TAT was better protected in the w/o microemulsion in an enzyme-containing environment, suggesting that the w/o microemulsions developed in this study may serve as a potential delivery vehicle for local intestinal delivery of peptides or proteins after oral administration.     

Resveratrol-Loaded Vesicular Elastic Nanocarriers Gel in Imiquimod-Induced Psoriasis Treatment: In Vitro and In Vivo Evaluation

This work aimed to develop a new efficient approach for safe treatment of psoriasis. To achieve that, resveratrol-loaded spanlastics(F1-F12) were prepared and evaluated by complete in vitro characterization. The two optimal formulations (F10 and F11) had their particle size in the nano range with high entrapment efficiency and sustainable drug release. These two formulae were incorporated in carbopol 934 gel formulations (G1-G8) with different concentrations of drug and carbopol 934 polymer. G1 and G5 (1% w/w Carbopol 934 gel and 0.1% resveratrol) showed 40.13% ± 2.017% and 73.76% ± 2.46%,8 hours drug release, respectively. Their pH was accepted and non-irritant. At a shear stress of 500 s^?1, G1 and G5 showed a reasonable viscosity of 1048.5 ± 2.12 cps and 954 ± 2.15 cps, respectively. In the in vivo psoriasis study, mice treated by G5 gel showed significant improvement of erythema and scaling compared to positive control group and they maintained healthy skin as shown in histopathological observations. Moreover, this group showed the least changes in mRNA expression of inflammatory cytokines. Concisely, our results suggest that selected carbopol gel of resveratrol-loaded spanlastics could maximize resveratrol topical anti-psoriatic effect.     

Ternary Complexes with Core-Shell Bilayer for Double Level Targeted Gene Delivery: In Vitro and In Vivo Evaluation

Purpose

Hyaluronic acid (HA)/polyethyleneimine-dexamethasone (PEI-Dex)/DNA ternary complexes with “core-shell” bilayer were developed for double level targeted gene delivery. A PEI₁₈₀₀-Dex, as a core, was applied to compact DNA into a nano-sized structure and facilitate the nuclear translocation of DNA after endocytosis into tumor cells, and a polyanion HA, as the outer corona, was employed to improve targeted tumor delivery and reduce cytotoxicity.

Methods

PEI-Dex was synthesized and characterized by ¹H NMR. The characterizations of ternary complexes were investigated. Their biological properties, including transfection efficiency, cytotoxicity, cellular uptake and in vivo efficacy were evaluated systemically.

Results

Ternary complexes with the size of about 160 nm exhibited the lowest cytotoxicity and the highest transfection efficiency in B16F10 cells among investigated complexes. The sub-cellular localization study confirmed that ternary complexes could facilitate more efficient cell uptake and nuclear transport of DNA than binary complexes. Moreover, Cy7-labeled ternary complexes obviously accumulated in the tumor after i.v. administration, indicating that ternary complexes could assist the DNA targeting to the tumor. In in vivo studies, HA/PEI₁₈₀₀-Dex/DNA ternary complexes showed confirmed anti-inflammation activity, and could significantly suppress tumor growth of tumor-bearing nude mice.

Conclusions

HA/PEI-Dex/DNA ternary complexes might be a promising targeted gene delivery system.     

Sustained Polymeric Delivery of Gene Silencing Antisense ODNs,siRNA, DNAzymes and Ribozymes: In Vitro and In Vivo Studies

Small interfering RNA (siRNA), antisense oligonucleotides (ODNs), ribozymes and DNAzymes have emerged as sequence-specific inhibitors of gene expression that may have therapeutic potential in the treatment of a wide range of diseases. Due to their rapid degradation in vivo, the efficacy of naked gene silencing nucleic acids is relatively short lived. The entrapment of these nucleic acids within biodegradable sustained-release delivery systems may improve their stability and reduce the doses required for efficacy. In this study, we have evaluated the potential in vitro and in vivo use of biodegradable poly (_d,_l-lactide-co-glycolide) copolymer (PLGA) microspheres as sustained delivery devices for ODNs, ribozyme, siRNA and DNA enzymes. In addition, we investigated the release of ODN conjugates bearing 5′-end lipophilic groups. The in vitro sustained release profiles of microsphere-entrapped nucleic acids were dependent on variables such as the type of nucleic acid used, the nature of the lipophilic group, and whether the nucleic acid used was single or double stranded. For in vivo studies, whole body autoradiography was used to monitor the bio-distribution of either free tritium-labelled ODN or that entrapped within PLGA microspheres following subcutaneous administration in Balb-c mice. The majority of the radioactivity associated with free ODN was eliminated within 24 h whereas polymer-released ODN persisted in organs and at the site of administration even after seven days post-administration. Polymer microsphere released ODN exhibited a similar tissue and cellular tropism to the free ODN. Micro-autoradiography analyses of the liver and kidneys showed similar bio-distribution for polymer-released and free ODNs with the majority of radioactivity being concentrated in the proximal convoluted tubules of the kidney and in the Kupffer cells of the liver. These findings suggest that biodegradable PLGA microspheres offer a method for improving the in vivo sustained delivery of gene silencing nucleic acids, and hence are worthy of further investigation as delivery systems for these macromolecules.     

Oral Delivery of Sodium Cromolyn: Preliminary Studies In Vivo and In Vitro

Purpose. Herein we report the discovery of a group of derivatized -amino acids that increase the oral bioavailability of sodium cromolyn. Methods. We prepared three N-acylated -amino acids and used these compounds to demonstrate the oral delivery of cromolyn in an in vivo rat model. In vitro experiments, including permeation studies and near infrared spectroscopy, were also performed to initiate an understanding of the mechanism by which these compounds facilitate cromolyn oral delivery. Results. Following oral administration to rats of solutions containing a combination of cromolyn and the delivery agent, significant systemic plasma concentrations of the drug were detected. In vitro studies suggest that absorption of the drug across the gastrointestinal membrane is a passive process. Conclusions. The absolute oral bioavailability of sodium cromolyn in the rat model is estimated to be ~5%. Preliminary mechanistic studies suggest that a complex of the cromolyn/delivery agent facilitates permeation across/through the membrane.     

Development of Liposomal Systems of Finasteride for Topical Applications: Design,Characterization, and In Vitro Evaluation

Finasteride (FNS) is a “drug of choice” for benign prostate hypertrophy and prostate cancer. The drug has also been reported to be useful orally in the treatment of some difficult-to-treat androgen-dependent skin disorders, such as seborrhea, acne, hirsutism, and androgenetic alopecia. However, the ideal route for its administration (i.e., topical) remains unexplored. This has logically suggested the search for strategic formulation approaches to make the drug effective on topical applications, hitherto unexplored. The present study targets the encasement of drug molecules in the interiors of vesicular compartments (liposomes) made up of hydrogenated phospholipids, as an attempt toward the development of a trans-epidermal therapeutic system of FNS. Multilamellar drug-loaded liposomes were prepared by thin-film hydration with sonication method and optimized with respect to drug payload, entrapment efficiency, and size by formulating different vesicular compositions under different process conditions. The vesicular systems consisting of saturated phospholipid (100 mg), cholesterol (50 mg), and FNS (5 mg) showed highest drug payload (2.9 mg/100 mg of total lipids), and drug entrapment efficiency (88.6%). Mean (± SD) vesicle size of the prepared liposomes was found to be 3.66 ± 1.6 μm. Significantly higher skin permeation of FNS through excised abdominal mice skin of FNS was achieved from the liposomal formulations vis-à-vis corresponding solution and conventional gels. Liposomal FNS formulations also showed more than fivefold higher deposition of drug in skin than the corresponding plain drug solution and conventional gel. Stability studies indicated that the liposomal formulations were quite stable in the refrigerated conditions for 2 months with negligible drug leakage or vesicle size alteration during the storage period. Results of the current studies with FNS-loaded vesicular systems project the high plausibility of a topical liposomal formulation for effective localized delivery of Finasteride.     

Inhibition of Rat Splenocyte Proliferation with Methylprednisolone: In Vivo Effect of Liposomal Formulation

The effect of a liposomal formulation of methylprednisolone (MPL) on the inhibition of lymphocyte proliferation in spleen cells was investigated following IV dosing in rats. Liposomes do not alter the suppressive action of MPL when placed in lymphocyte culture. Rat splenocytes were found to have greater sensitivity to MPL (EC₅₀ = 7.9 nM) than do human peripheral blood lymphocytes (EC₅₀ = 28 nM). In vivo studies in rats utilized 2 mg/kg IV bolus doses of liposomal MPL compared to drug in solution. Animals were sacrificed at various times post-dosing until 120 h, spleen was excised and, after incubation of lymphocytes with PHA, splenocyte blastogenic responses were assessed by measuring cellular incorporation of ³H-thymidine. The suppressive effect of liposomal MPL in comparison with free drug was significantly prolonged (>120 h vs < 18 h). Inhibition effects versus time were described by a pharmacodynamic model using MPL concentrations in plasma as an input function. A nonlinear relationship was found between suppression of splenocyte proliferation and the concentration of bound glucocorticoid receptors in spleen. Only partial receptor occupancy accompanied complete lymphocyte suppression. The suppression of endogenous corticosterone in plasma for both treatments was similar with values from L-MPL rats returning to baseline after 24 h. These results demonstrate enhanced efficacy of local immunosuppression by targeting spleen with liposomal MPL.