Microfluidic Synthesis of PEG- and Folate-Conjugated Liposomes for One-Step Formation of Targeted Stealth Nanocarriers

Purpose

A microfluidic hydrodynamic flow focusing technique enabling the formation of small and nearly monodisperse liposomes is investigated for continuous-flow synthesis of poly(ethylene glycol) (PEG)-modified and PEG-folate-functionalized liposomes for targeted drug delivery.

Methods

Controlled laminar flow in thermoplastic microfluidic devices facilitated liposome self-assembly from initial lipid compositions including lipid/cholesterol mixtures containing PEG-lipid and folate-PEG-lipid conjugates. Relationships among flow conditions, lipid composition, and liposome size were evaluated; their impact on PEG and folate incorporation were determined through a combination of UV–vis absorbance measurements and characterization of liposome zeta potential.

Results

PEG and folate were successfully incorporated into microfluidic-synthesized liposomes over the full range of liposome sizes studied. Efficiency of PEG-lipid incorporation was inversely correlated with liposome diameter. Folate-lipid was effectively integrated into liposomes at various flow conditions.

Conclusions

Liposomes incorporating relatively large PEG-modified and folate-PEG-modified lipids were successfully synthesized using the microfluidic flow focusing platform, providing a simple, low cost, rapid method for preparing functionalized liposomes. Relationships between preparation conditions and PEG or folate-PEG functionalization have been elucidated, providing insight into the process and defining paths for optimization of the microfluidic method toward the formation of functionalized liposomes for pharmaceutical applications.     

Rapid Lymph Accumulation of Polystyrene Nanoparticles Following Pulmonary Administration

Purpose

Pulmonary administration of polymeric nanoparticle drug delivery systems is of great interest for both systemic and local therapies. However, little is understood about the relationship of particle size and pulmonary absorption. We investigated uptake and biodistribution of polystyrene nanoparticles (PN) of 50 nm, 100 nm, 250 nm, and 900 nm diameters in mice following administration to lungs via pharyngeal aspiration.

Methods

The amount of PN in tissues was analyzed by gel permeation chromatography (GPC).

Results

At 1 h, larger diameter PN (250 nm and 900 nm) had the highest total uptake at around 15% of administered dose, whereas the smaller diameter PN (50 nm and 100 nm) had uptake of only 5–6%. However, at 3 h, the 50 nm PN had the highest total uptake at 24.4%. For each size tested, the highest nanoparticle deposition was observed in the lymph nodes (LN) as compared to other tissues accounting for a total of about 35–50% of absorbed nanoparticles.

Conclusion

PN size impacts the rate and extent of uptake from lungs and, further, the extent of LN deposition. The extent of uptake and lymph distribution of the model, non-degradable PN lends potential to pulmonary administered, biodegradable polymeric nanoparticles for delivery of therapeutics to regional lymph nodes.     

Chitooligosaccharides Modified Reduction-Sensitive Liposomes: Enhanced Cytoplasmic Drug Delivery and Osteosarcomas-Tumor Inhibition in Animal Models

Purpose

To investigate the potential of a reduction-sensitive and fusogenic liposomes, enabled by surface-coating with chotooligosaccharides (COS) via a disulfide linker, for tumor-targeted cytoplasmic drug delivery.

Methods

COS (MW2000-5000) were chemically tethered onto the liposomes through a disulfide linker (-SS-) to cholesterol (Chol). Doxorubicin (DOX) was actively loaded in the liposomes. Their reduction-sensitivities, cellular uptake, cytotoxicity, pharmacokinetics and antitumor efficacy were investigated.

Results

The Chol-SS-COS/DOX liposomes (100 nm) had zeta potential of 33.9 mV and high drug loading (13% w/w). The liposomes were stable with minimal drug leakage under physiological conditions but destabilized in the presence of reducing agents, dithiothreitol (DTT) or glutathione (GSH) at 10 mM, the cytosolic level. MTT assay revealed that the cationic Chol-SS-COS/DOX liposomes had higher cytotoxicity to MG63-osteosarcoma cells than non-reduction sensitive liposome (Chol-COS/DOX). Flow cytometry and confocal microscopy revealed that Chol-SS-COS/DOX internalized more efficiently than Chol-COS/DOX with more content to cytoplasm whereas Chol-COS/DOX located around the cell membrane. Chol-SS-COS/DOX preferentially internalized into MG63 cancer cell over LO2 normal liver cells. In rats both liposomes produced a prolonged half-life of DOX by 4 - 5.5 fold (p < 0.001) compared with the DOX solution. Chol-SS-COS/DOX exhibited strong inhibitory effect on tumor growth in MG63 cell-bearing nude mice (n = 6), and extended animal survival rate.

Conclusions

Reduction-responsive Chol-SS-COS liposomes may be an excellent platform for cytoplasmic delivery of anticancer drugs. Conjugation of liposomes with COS enhanced tumor cell uptake, antitumor effect and survival rate in animal models.

     

Liposomal Aerosols of Nitric Oxide (NO) Donor as a Long-Acting Substitute for the Ultra-Short-Acting Inhaled NO in the Treatment of PAH

Purpose

This study seeks to develop a liposomal formulation of diethylenetriamine NONOate (DN), a long acting nitric oxide (NO) donor, with a goal to replace inhaled NO (iNO) in the treatment of pulmonary arterial hypertension (PAH).

Methods

Liposomal formulations were prepared by a lipid film hydration method and modified with a cell penetrating peptide, CAR. The particles were characterized for size, polydispersity index (PDI), zeta potential, entrapment efficiency, storage and nebulization stability, and in-vitro release profiles. The cellular uptake and transport were assessed in rat alveolar macrophages (NR8383) and transforming growth factor β (TGF-β) activated rat pulmonary arterial smooth muscle cells (PASMCs). The fraction of the formulation that enters the systemic circulation, after intratracheal administration, was determined in an Isolated Perfused Rat Lung (IPRL) model. The safety of the formulations were assessed using an MTT assay and by measuring injury markers in the bronchoalveolar lavage (BAL) fluid; the pharmacological efficacy was evaluated by monitoring the changes in the mean pulmonary arterial (mPAP) and systemic pressure (mSAP) in a monocrotaline (MCT) induced-PAH rat model

Results

Liposome size, zeta potential, and entrapment efficiency were 171?±?4 nm, -37?±?3 mV, and 46?±?5%, respectively. The liposomes released 70?±?5% of the drug in 8 h and were stable when stored at 4°C. CAR-conjugated-liposomes were taken up more efficiently by PASMCs than liposomes-without-CAR; the uptake of the formulations by rat alveolar macrophages was minimal. DN-liposomes did not increase lung weight, protein quantity, and levels of injury markers in the BAL fluid. Intratracheal CAR-liposomes reduced the entry of liposomes from the lung to blood; the formulations produced a 40% reduction in mPAP for 180 minutes.

Conclusion

This study establishes the proof-of-concept that peptide modified liposomal formulations of long-acting NO donor can be an alternative to short-acting iNO.

     

The Potential Efficacy of R8-Modified Paclitaxel-Loaded Liposomes on Pulmonary Arterial Hypertension

Purpose

In this paper, a novel liposomal formulation of paclitaxel modified with octaarginine (R8) was fabricated and the therapeutic efficacy of it on pulmonary arterial hypertension was evaluated.

Methods

Octaarginine-modified stealth liposomes loaded with PTX (R8-PTX-LIP) were prepared and characterized. Vector cytoxicity and anti-proliferation ability of different formulations on primary cultured VSMCs were determined with MTT assay. The uptake capacity of VSMCs on different formulations were evaluated by flow cytometry, and the influences on cytoskeletons of liposomes were investigated by cytoskeleton staining with rhodamine-phalloidin. The biodistribution of liposomes were imaged by a CCD camera using a near-infrared fluorophore DiD. The therapeutic efficacy of different PTX-formulations of PAH was evaluated by hemodynamic measurement, right ventricular hypertrophic parameters and vessel diameters.

Results

The cellular uptake of R8 modified liposomes (R8-LIP) was improved noticeably compared with other groups. All liposomes did not exert cytotoxicity on VSMCs in 24 h. R8-PTX-LIP exhibited the strongest inhibitory effect on the proliferation of VSMCs among all the formulations (p?<?0.001). R8-PTX-LIP could reverse the phenotype transformation, and inhibit cell migration. mPAP, (RV/LV+S) and the wall thickness of small distal pulmonary arteries of rats treated with R8-PTX-LIP were significantly lower than those from other groups (p?<?0.001).

Conclusions

In conclusion, the drug delivery system of R8-modified paclitaxel-loaded liposomes we established showed pronounced inhibitory effect over VSMCs proliferation and cytoskeleton formation in vitro, a stronger pulmonary delivery ability in vivo, and was effective on PAH, showing the potential for pulmonary drug delivery system for PAH treatment.     

Development of High-Content Gemcitabine PEGylated Liposomes and Their Cytotoxicity on Drug-Resistant Pancreatic Tumour Cells

Purpose

The objective of this study was to develop high-content gemcitabine PEGylated liposomes to reverse gemcitabine resistance in pancreatic tumour cells. The mechanism of drug loading into liposomes was also investigated.

Methods

To increase the drug entrapment efficiency (EE) and drug loading (DL), a novel passive loading approach named Small Volume Incubation method (SVI) was developed and compared to the reverse phase evaporation (REV) and remote loading methods. The in vitro cytotoxicity was evaluated using MIA PaCa-2 and Panc-1 cell lines.

Results

The EE for remote loading was 12.3?±?0.3%, much lower than expected and a burst release was observed with the resultant liposomes. Using the optimized SVI method, increased EE (37?±?1%) and DL (4%, w/w) were obtained. The liposomes (200?±?5 nm) showed minimal drug leakage, good stability, and significant improvement in cytotoxicity to the gemcitabine-resistant pancreatic cancer cell lines.

Conclusions

Remote loading was not suitable for loading gemcitabine into liposomes. pK_a?>?4.6 for basic drugs and intra-liposomal precipitation of loaded compounds were suggested as an additional requirement to the current criteria for remote loading using ammonium sulphate gradient (pK_a?相似文献   

The Bifunctional Liposomes Constructed by Poly(2-ethyl-oxazoline)-cholesteryl Methyl Carbonate: an Effectual Approach to Enhance Liposomal Circulation Time,pH-Sensitivity and Endosomal Escape

Purpose

A novel bifunctional liposome with long-circulating and pH-sensitive properties was constructed using poly(2-ethyl-oxazoline)-cholesteryl methyl carbonate (PEtOz-CHMC) in this study.

Methods

PEtOz-CHMC was synthesized and characterized by TLC, IR and ¹H-NMR. The obtained PEtOz lipid was inserted into liposomes by the post-insertion method. Through a series of experiments, such as drug release, tumor cell uptake, cytotoxicity, calcium-induced aggregation, pharmacokinetic experiments, etc., the pH-sensitive and long-circulating properties of PEtOzylated liposomes was identified.

Results

PEtOz-CHMC modified liposomes (PEtOz-L) showed increased calcein release at low pH. Flow cytometric analysis results showed that the fusion and cellular uptake of PEtOz-L could be promoted significantly at pH 6.4 compared with those at pH 7.4. Confocal laser scanning microscope observations revealed that PEtOz-L could respond to low endosomal pH and directly released the fluorescent tracer into the cytoplasm. MTT assays in HeLa cells demonstrated that doxorubicin hydrochloride (DOX) loaded PEtOz-L exhibited stronger anti-tumor activity in a medium at pH 6.4 than in a medium pH 7.4. PEtOz-L remained stable when these liposomes were incubated in calcium chloride solution. The cumulative calcein release rate of PEtOz-L was significantly lower than that of CL when the liposomes were dialysed in PBS. The pharmacokinetic experiments of liposomes in rats showed that t _1/2 and AUC of PEtOz-L were 4.13 times and 4.71 times higher than those of CL.

Conclusions

PEtOzylated liposomes exhibits excellent long-circulating and pH-sensitive properties. Our results suggest that PEtOz is a promising biomaterial for the modification of liposome in drug delivery.     

Transferrin-Modified Vitamin-E/Lipid Based Polymeric Micelles for Improved Tumor Targeting and Anticancer Effect of Curcumin

Purpose

Transferrin receptor (TfR) is up-regulated in various malignant tumors not only to meet the iron requirement, but also to increase the cell survival via participation in various cellular signaling pathways. Here we explored transferrin as ligand for Poly(ethylene Glycol) (PEG)-ylated vitamin-E/lipid (PE) core micelles (VPM).

Methods

Transferrin modified polymer was synthesized and drug loaded micelles were evaluated in 2D Hela and HepG2 cancer cells for cellular uptake and cytotoxicity and in 3D Hela spheroids for growth inhibition, uptake and penetration studies.

Results

Targeted (Tf-VPM) and non-targeted (VPM) micelles showed mean hydrodynamic diameter of 114.2?±?0.64 nm and 117.4?±?0.72 nm and zeta potential was ?22.8?±?0.62 and ?14.8?±?1.74 mV, respectively. Cellular uptake study indicated that the Tf-CVPM were taken up by cancer cells (Hela and HepG2) with higher efficiency. Enhanced cytotoxicity was demonstrated for Tf-VPM compared to CVPM. Marked spheroid growth inhibition following treatment with Tf-CVPM was observed compared to the treatment with non-targeted CVPM.

Conclusions

The developed transferrin-modified micelles have improved ability to solubilize the loaded drugs and could actively target solid tumors by its interaction with over-expressed transferrin receptors. Therefore, the nano-micelles could be further explored for its potential utilization in cancer therapy.

     

Biodistribution,Tumor Uptake and Efficacy of 5-FU-Loaded Liposomes: Why Size Matters

Purpose

We have investigated the impact of particle size on the biodistribution, tumor uptake and antiproliferative efficacy of 5-FU-loaded liposomes.

Methods

Three different batches of pegylated liposomes varying in size (i.e., 70, 120 and 250 nm respectively) were tested. The active compounds encapsulated were an equimolar mix of 5-FU, 2′-deoxyinosine and folinic acid. Liposomes were subsequently tested on the human breast cancer model MDA231 cells, a model previously found to be resistant to 5-FU. In vitro, antiproliferative efficacy and microscopy studies of liposomes uptake were carried out. In vivo, comparative biodistribution and efficacy studies were performed in tumor-bearing mice.

Results

Difference in size did not change in vitro antiproliferative activity. Fluorescence-Microscopy studies showed that liposomes were mainly uptaken by tumor cells through a direct internalization process, regardless of their size. Biodistribution profiles in tumor-bearing mice revealed higher accumulation of small liposomes in tumors throughout time as compared with normal and large liposomes (p?in vivo efficacy studies showed at study conclusion that a 68% reduction in tumor size was achieved with small liposomes (p?Conclusion This study suggests that particle size is critical to achieve higher selectivity and efficacy in experimental oncology, including in resistant tumors.     

Folate-Targeted Nanoparticles Based on Albumin and Albumin/Alginate Mixtures as Controlled Release Systems of Tamoxifen: Synthesis and In Vitro Characterization

Purpose

Preparation and in vitro characterization of tamoxifen (TMX)-loaded folate-targeted nanoparticles based on disulfide bond reduced bovine serum albumin (BSA-SH) and BSA-SH/alginate-cysteine (BSA-SH/ALG-CYS) mixtures as drug delivery systems.

Methods

Folate-nanoparticles were characterized in terms of folate content, morphology, size, zeta potential, TMX load and drug release kinetics. Additionally, cell viability and cellular uptake of nanoparticles were determined using different cancer cell lines.

Results

Folic acid (FOL) was successfully attached to nanoparticles (ranging between 79 and170 μmol folate/g NP). Nanoparticles with 76–417 nm mean size were obtained and loaded with TMX (4.2–7.7 μg/mg NP). Zeta potential and drug extraction revealed major superficial placement of the drug, especially in the case of BSA/ALG-FOL systems. Drug release studies in the presence of surfactant showed a gradual release of the drug between 4–7 h. In general, low cytotoxicity of unloaded systems was found. Internalization of the systems was achieved and mediated by folate receptor, especially in the case of BSA NP-FOL. The administration of 10 μM TMX by TMX-FOL NP showed their efficacy as controlled TMX release systems.

Conclusions

Promising anticancer action of these new TMX-loaded folate-targeted systems was demonstrated, allowing a new administration route to be studied in further in vivo studies in order to improve current TMX therapy.     

Sterically Stabilized Liposomes Incorporating the Novel Anticancer Agent Phospho-Ibuprofen (MDC-917): Preparation, Characterization, and In Vitro/In Vivo Evaluation

Purpose

To incorporate phospho-ibuprofen (P-I), a lipophilic, water insoluble novel anti-cancer agent, into pegylated liposomes and upon formulation optimization to evaluate its antitumor activity in vitro and in vivo.

Methods

P-I loaded liposomes were prepared using the thin-film hydration method, and characterized for size, zeta potential, drug content and drug release. We examined their physical stability by particle size changes; their lyophilization ability in the presence of cryoprotectants; and their antitumor activity in vitro in human cancer cell lines and in vivo in a xenograft murine model.

Results

P-I was successfully loaded into liposomes consisting of soy-PC and PEG₂₀₀₀-PE. These liposomes were <150?nm in diameter; exhibited prolonged stability in suspension and can be lyophilized using sucrose as cryoprotectant. P-I liposomes inhibited the growth of human cancer cell lines in vitro and in vivo of xenograft in nude mice to a greater extent than free P-I.

Conclusions

High levels of P-I can be incorporated into liposomes which can be lyophilized in the presence of sucrose and showed good stability upon storage. Moreover, these drug-incorporating liposomes were capable of inhibiting the growth of xenografted tumors in mice more effectively than free P-I. These results justify further development of the P-I liposomes.     

Near-Infrared Image-Guided Delivery and Controlled Release Using Optimized Thermosensitive Liposomes

Purpose

To engineer optimized near-infrared (NIR) active thermosensitive liposomes to potentially achieve image-guided delivery of chemotherapeutic agents.

Methods

Thermosensitive liposomes were surface-coated with either polyethylene glycol or dextran. Differential scanning calorimetry and calcein release studies were conducted to optimize liposomal release, and flow cytometry was employed to determine the in vitro macrophage uptake of liposomes. Indocyanine green (ICG) was encapsulated as the NIR dye to evaluate the in vivo biodistribution in tumor-bearing mice.

Results

The optimized thermosensitive liposome formulation consists of DPPC, SoyPC, and cholesterol in the 100:50:30 molar ratio. Liposomes with dextran and polyethylene glycol demonstrated similar thermal release properties; however in vitro macrophage uptake was greater with dextran. Non-invasive in vivo NIR imaging showed tumor accumulation of liposomes with both coatings, and ex vivo NIR imaging correlated well with actual ICG concentrations in various organs of healthy mice.

Conclusions

The optimized thermosensitive liposome formulation demonstrated stability at 37?°C and efficient burst release at 40 and 42?°C. Dextran exhibited potential for application as a surface coating in thermosensitive liposome formulations. In vivo studies suggest that liposomal encapsulation of ICG permits reliable, real-time monitoring of liposome biodistribution through non-invasive NIR imaging.     

Nano-Engineered Erythrocyte Ghosts as Inhalational Carriers for Delivery of Fasudil: Preparation and Characterization

Purpose

Nanoerythrosomes (NERs), an engineered derivative of erythrocytes, have long been used as drug delivery carriers. These cell based carriers are biocompatible and biodegradable, and they exhibit efficient drug loading, targeting specificity and prolonged biological half-life. In this study, we have evaluated the feasibility of NERs as inhalable carriers for delivery of fasudil, an investigational drug for the treatment of pulmonary arterial hypertension.

Methods

We prepared NERs by hypotonic lysis of erythrocytes derived from rat blood followed by extrusion through polycarbonate membranes. The formulations were optimized and characterized for size, morphology, entrapment efficiency, stability, cellular uptake and in-vitro release profiles followed by monitoring of drug absorption and safety evaluation after intratracheal administration of fasudil-loaded NERs into rats.

Results

NERs were spherical in shape with an average size of 154.1?±?1.31 nm and the drug loading efficiency was 48.76?±?2.18%. Formulations were stable when stored at 4°C for 3 weeks. When incubated with rat pulmonary arterial smooth muscle cells (PASM), a significant amount of NERs was taken up by PASM cells. The drug encapsulated in NERs inhibited the rho-kinase activity upto 50%, which was comparable with the plain fasudil. A ~6–8 fold increase in the half-life of fasudil was observed when encapsulated in NERs.

Conclusion

This study suggests that nanoerythrosomes can be used as cell derived carriers for inhalational delivery of fasudil.     

Characterization of Spherulites as a Lipidic Carrier for Low and High Molecular Weight Agents

Purpose

To develop spherulite formulations to achieve high entrapment efficiency for both small and macromolecules as well as cell-type specific delivery.

Methods

Spherulites of various compositions were prepared, and lipid-PEG was incorporated through post-insertion. Calcein and FITC-labeled albumin were employed as model drugs for small and macromolecules. The spherulites were characterized with respect to entrapment efficiency, size, structure, and release kinetics, and the morphology was examined via cryo-EM. Finally, SV119-decorated spherulites were examined for their selective uptake by cancer cells.

Results

The spherulites are 170 ~ 290 nm in size. A loading efficiency of 55 ~ 60% can be consistently achieved for both calcein and albumin under optimized conditions. Cryo-EM shows the onion-like morphology consistent with the structure of multilamellar liposomes. A t_1/2 of 39.3 h and 69.7 h in cargo release in serum was observed before and after PEG decoration, and incorporation of SV119 led to selective delivery of rhodamine-labeled spherulites to PC-3 tumor cells.

Conclusions

Our optimized formulations may represent a platform with simple preparation approach, relatively small particle size, high drug loading efficiency for both low and high molecular weight agents, and slow release kinetics for selective delivery of various types of therapeutics to target cells.     

EphA2 Targeting Pegylated Nanocarrier Drug Delivery System for Treatment of Lung Cancer

Purpose

Evaluation of tumor targeting pegylated EphA2 peptide coated nanoparticles (ENDDs) of a novel anticancer agent DIM-C-pPhC₆H₅ (DIM-P) and Docetaxel (DOC) and investigate its antitumor activity and potential for treatment of lung cancer.

Methods

Nanoparticles were prepared with DIM-P and DOC (NDDs) using Nano-DeBEE. ENDDs were prepared by conjugating NDDs with 6His-PEG2K-EphA2 peptide and characterized for physicochemical properties, binding assay, cytotoxicity, cellular uptake studies, drug release and pharmacokinetic parameters. Anti-tumor activity of ENDDs was evaluated using a metastatic H1650 and orthotopic A549 tumor models in nude mice and tumor tissue were analyzed by RT-PCR and immunohistochemistry.

Results

Particle size and entrapment efficiency of ENDDs were 197?±?21 nm and 95?±?2%. ENDDs showed 32.5?±?3.5% more cellular uptake than NDDs in tumor cells. ENDDs showed 23?±?3% and 26?±?4% more tumor reduction compared to NDDs in metastatic and orthotopic tumor models, respectively. In-vivo imaging studies using the Care stream MX FX Pro system showed (p?Conclusions The results emanating from these studies demonstrate anti-cancer potential of DIM-P and the role of ENDDs as effective tumor targeting drug delivery systems for lung cancer treatment.     

Surface Analysis of PEGylated Nano-Shields on Nanoparticles Installed by Hydrophobic Anchors

Purpose

This work describes a method for functionalisation of nanoparticle surfaces with hydrophilic “nano-shields” and the application of advanced surface characterisation to determine PEG amount and accumulation at the outmost 10 nm surface that is the predominant factor in determining protein and cellular interactions.

Methods

Poly(lactic-co-glycolic acid) (PLGA) nanoparticles were prepared with a hydrophilic PEGylated “nano-shield” inserted at different levels by hydrophobic anchoring using either a phospholipid-PEG conjugate or the copolymer PLGA-block-PEG by an emulsification/diffusion method. Surface and bulk analysis was performed including X-ray photoelectron spectroscopy (XPS), nuclear magnetic resonance spectroscopy (NMR) and zeta potential. Cellular uptake was investigated in RAW 264.7 macrophages by flow cytometry.

Results

Sub-micron nanoparticles were formed and the combination of (NMR) and XPS revealed increasing PEG levels at the particle surface at higher PLGA-b-PEG copolymer levels. Reduced cellular interaction with RAW 264.7 cells was demonstrated that correlated with greater surface presentation of PEG.

Conclusion

This work demonstrates a versatile procedure for decorating nanoparticle surfaces with hydrophilic “nano-shields”. XPS in combination with NMR enabled precise determination of PEG at the outmost surface to predict and optimize the biological performance of nanoparticle-based drug delivery.     

Influence of Short-Chain Cell-Penetrating Peptides on Transport of Doxorubicin Encapsulating Receptor-Targeted Liposomes Across Brain Endothelial Barrier

Purpose

To investigate the influence of different cell penetrating peptides (CPPs-TAT, Penetratin and Mastoparan), on the transport of doxorubicin encapsulating transferrin (Tf)-liposomes across brain endothelial barrier, in vitro and in vivo.

Methods

The cellular uptake of dual-functionalized, (Tf-CPP), liposomes into various tumor cells was assessed using HPLC. The transport of liposomes was also measured across a robust 3D brain tumor model constructed using chitosan-PLGA scaffolds. The growth of tumor cells was monitored using H&E staining and the fully grown tumor scaffolds were visualized using SEM. The tumor scaffolds were combined with the culture inserts carrying tightly packed brain endothelial cells. The in vitro and in vivo transport of drug (using Tf-CPP-liposomes) across the brain endothelial barrier was determined by extraction of the drug from cells and tissues followed by analysis using HPLC.

Results

The results demonstrated improved delivery of doxorubicin using dual-functionalized liposomes versus the single ligand or unmodified liposomes. Among different Tf-CPP-liposomes, the Tf-Penetratin liposomes showed efficient cellular transport of the encapsulated drug (approximately 90–98%) and maximum translocation of the drug across the brain endothelial barrier (approximately 15% across in vitro and 4% across in vivo BBB). The Tf-Penetratin and Tf-TAT liposomes demonstrated excellent cellular biocompatibility and no hemolytic activity upto 200nM phospholipid concentration.

Conclusions

The Tf-CPP liposomes showed efficient translocation of the anticancer drug across the brain endothelial barrier. In addition, an absolute and robust in vitro brain tumor model was successfully constructed to overcome the practical intricacies of developing a successful in vivo orthotopic brain tumor model.     

Gemcitabine Treatment of Rat Soft Tissue Sarcoma with Phosphatidyldiglycerol-Based Thermosensitive Liposomes

Purpose

The pyrimidine analogue gemcitabine (dFdC) is frequently used in the treatment of patients with solid tumors. However, after i.v. application dFdC is rapidly inactivated by metabolization. Here, the potential of thermosensitive liposomes based on 1,2-dipalmitoyl-sn-glycero-3-phosphodiglycerol (DPPG₂-TSL) were investigated as carrier and targeting system for delivery of dFdC in combination with local hyperthermia (HT).

Methods

DPPG₂-TSL were prepared by the lipid film hydration and extrusion method and characterized by dynamic light scattering, thin layer chromatography, phosphate assay and HPLC. In vivo experiments were performed in Brown Norway rats with a syngeneic soft tissue sarcoma. Local HT treatment was performed by light exposure.

Results

DPPG₂-TSL were stable at 37°C in serum and showed a temperature dependent dFdC release >40°C. Plasma half-life of dFdC was strongly increased from 0.07 h (non-liposomal) to 0.53 h (liposomal, vesicle size 105 nm) or 2.59 h (liposomal, 129 nm). Therapy of BN175 tumors with dFdC encapsulated in DPPG₂-TSL + HT showed significant improvement in tumor growth delay compared to non-liposomal dFdC without HT (p?p?p?Conclusions Gemcitabine encapsulated in DPPG₂-TSL in combination with local HT is a promising tool for the treatment of solid tumors. Therefore, these encouraging results ask for further investigation and evaluation.     

Effects of Particle Size and Surface Modification on Cellular Uptake and Biodistribution of Polymeric Nanoparticles for Drug Delivery

Purpose

To investigate the effects of the particle size and surface coating on the cellular uptake of the polymeric nanoparticles for drug delivery across the physiological drug barrier with emphasis on the gastrointestinal (GI) barrier for oral chemotherapy and the blood–brain barrier (BBB) for imaging and therapy of brain cancer.

Methods

Various sizes of commercial fluorescent polystyrene nanoparticles (PS NPs) (viz 20 50, 100, 200 and 500 nm) were modified with the d-α-tocopheryl polyethylene glycol 1,000 succinate (vitamin E TPGS or TPGS). The size, surface charge and surface morphology of PS NPs before and after TPGS modification were characterized. The Caco-2 and MDCK cells were employed as an in vitro model of the GI barrier for oral and the BBB for drug delivery into the central nerve system respectively. The distribution of fluorescent NPs after i.v. administration to rats was analyzed by the high performance liquid chromatography (HPLC).

Results

The in vitro investigation showed enhanced cellular uptake efficiency for PS NPs in both of Caco-2 and MDCK cells after TPGS surface coating. In vivo investigation showed that the particle size and surface coating are the two parameters which can dramatically influence the NPs biodistribution after intravenous administration. The TPGS coated NPs of smaller size (< 200 nm) can escape from recognition by the reticuloendothelial system (RES) and thus prolong the half-life of the NPs in the blood system.

Conclusions

TPGS-coated PS NPs of 100 and 200 nm sizes have potential to deliver the drug across the GI barrier and the BBB.     

Lysozyme Release and Polymer Erosion Behavior of Injectable Implants Prepared from PLGA-PEG Block Copolymers and PLGA/PLGA-PEG Blends

Purpose

We evaluated the controlled release of lysozyme from various poly(D,L-lactic-co-glycolic acid) (PLGA) 50/50-polyethylene glycol (PEG) block copolymers relative to PLGA 50/50.

Methods

Lysozyme was encapsulated in cylindrical implants (0.8 mm diameter) by a solvent extrusion method. Release studies were conducted in phosphate buffered saline +0.02% Tween 80 (PBST) at 37°C. Lysozyme activity was measured by a fluorescence-based assay. Implant erosion was evaluated by kinetics of polymer molecular weight decline, water uptake, and mass loss.

Results

Lysozyme release from an AB15 di-block copolymer (15% 5 kDa PEG, PLGA 28 kDa) was very fast, whereas an AB10 di-block copolymer (with 10% 5 kDa PEG, PLGA 45 kDa) and ABA10 tri-block copolymer (with 10% 6 kDa PEG, PLGA 27 kDa) showed release profiles similar to PLGA. We achieved continuous lysozyme release for up to 4 weeks from AB10 and ABA10 by lysozyme co-encapsulation with the pore-forming and acid-neutralizing MgCO₃, and from AB15 by co-encapsulation of MgCO₃ and blending AB15 with PLGA. Lysozyme activity was mostly recovered during 4 weeks.

Conclusions

These block co-polymers may have utility either alone or as PLGA blends for the controlled release of proteins.