Near-Infrared Light-Sensitive Liposomes for the Enhanced Photothermal Tumor Treatment by the Combination with Chemotherapy

Purpose

To develop a near-infrared (NIR) light-sensitive liposome, which contains hollow gold nanospheres (HAuNS) and doxorubicin (DOX), and evaluate their potential utility for enhancing antitumor activity and controlling drug release.

Methods

The liposomes (DOX&HAuNS-TSL) were designed based on a thermal sensitive liposome (TSL) formulation, and hydrophobically modified HAuNS were attached onto the membrane of the liposomes. The behavior of DOX release from the liposomes was investigated by the dialysis, diffusion in agarose gel and cellular uptake of the drug. The biodistribution of DOX&HAuNS-TSL was assessed by i.v. injection in tumor-bearing nude mice. Antitumor efficacy was evaluated both histologically using excised tissue and intuitively by measuring the tumor size and weight.

Results

Rapid and repetitive DOX release from the liposomes (DOX&HAuNS-TSL), could be readily achieved upon NIR laser irradiation. The treatment of tumor cells with DOX&HAuNS-TSL followed by NIR laser irradiation showed significantly greater cytotoxicity than the treatment with DOX&HAuNS-TSL alone, DOX-TSL alone (chemotherapy alone) and HAuNS-TSL plus NIR laser irradiation (Photothermal ablation, PTA, alone). In vivo antitumor study indicated that the combination of simultaneous photothermal and chemotherapeutic effect mediated by DOX&HAuNS-TSL plus NIR laser presented a significantly higher antitumor efficacy than the PTA alone mediated by HAuNS-TSL plus NIR laser irradiation.

Conclusions

Our study could be as the valuable reference and direction for the clinical application of PTA in tumor therapy.     

PLGA/Liposome Hybrid Nanoparticles for Short-Chain Ceramide Delivery

Purpose

Rapid premature release of lipophilic drugs from liposomal lipid bilayer to plasma proteins and biological membranes is a challenge for targeted drug delivery. The purpose of this study is to reduce premature release of lipophilic short-chain ceramides by encapsulating ceramides into liposomal aqueous interior with the aid of poly (lactic-coglycolicacid) (PLGA).

Methods

BODIPY FL labeled ceramide (FL-ceramide) and BODIPY-TR labeled ceramide (TR-ceramide) were encapsulated into carboxy-terminated PLGA nanoparticles. The negatively charged PLGA nanoparticles were then encapsulated into cationic liposomes to obtain PLGA/liposome hybrids. As a control, FL-ceramide and/or TR ceramide co-loaded liposomes without PLGA were prepared. The release of ceramides from PLGA/liposome hybrids and liposomes in rat plasma, cultured MDA-MB-231 cells, and rat blood circulation was compared using fluorescence resonance energy transfer (FRET) between FL-ceramide (donor) and TR-ceramide (acceptor).

Results

FRET analysis showed that FL-ceramide and TR-ceramide in liposomal lipid bilayer were rapidly released during incubation with rat plasma. In contrast, the FL-ceramide and TR-ceramide in PLGA/liposome hybrids showed extended release. FRET images of cells revealed that ceramides in liposomal bilayer were rapidly transferred to cell membranes. In contrast, ceramides in PLGA/liposome hybrids were internalized into cells with nanoparticles simultaneously. Upon intravenous administration to rats, ceramides encapsulated in liposomal bilayer were completely released in 2 min. In contrast, ceramides encapsulated in the PLGA core were retained in PLGA/liposome hybrids for 4 h.

Conclusions

The PLGA/liposome hybrid nanoparticles reduced in vitro and in vivo premature release of ceramides and offer a viable platform for targeted delivery of lipophilic drugs.     

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.     

Multi-functional Liposomes Enhancing Target and Antibacterial Immunity for Antimicrobial and Anti-Biofilm Against Methicillin-Resistant <Emphasis Type="Italic">Staphylococcus aureus</Emphasis>

Purpose

The aim of this study was to prepare wheat germ agglutinin (WGA)-modified liposomes encapsulating clarithromycin and to evaluate their in vitro and in vivo efficacy against Methicillin-resistant Staphylococcus aureus (MRSA).

Methods

Physicochemical parameters, minimum inhibitory concentrations, in vitro killing kinetic, cellular uptake, biofilm formation inhibition and pre-formed biofilm destruction, biodistribution, in vivo antibacterial efficacy against MRSA, and phagocytosis into macrophages for liposomes loading clarithromycin were determined.

Results

The minimum inhibitory concentration and the time–kill curve for WGA-modified liposomal clarithromycin were better than those of free and nonmodified liposomal clarithromycin. Flow cytometry analysis displayed that liposomes could deliver more Coumarin 6, a fluorescent probe, into bacteria because of the conjugation of WGA. Besides, WGA-modified liposomal clarithromycin inhibited formation of S. aureus (ATCC 29213) and MRSA biofiom, and prompted the biofilm disassembly at lower concentrations below MIC. Effective accumulation of liposomes was displayed in the enterocoelia of the mice because of WGA. The number of MRSA colony-forming units in the kidney and spleen in mice treated with WGA-modified liposomal clarithromycin was significantly lower than that treated with free and nonmodified clarithromycin (p?<?0.05). Intracellular localization of MRSA occurred in a significantly higher proportion of macrophage exposed to WGA-modified liposomes compared to those exposed to nonmodified liposomes.

Conclusions

Liposome modified by WGA is a promising formulation for bacteria targeted delivery and immunity defensive system through macrophage improving uptake of bacteria, biodistribution, in vitro and in vivo antibacterial efficacy against MRSA.

     

Brain-specific delivery of rifampin from lactyl stearate-coupled liposomes via monocarboxylic acid transporters

Background

The blood-brain barrier (BBB) is an obstacle for pharmacologists wishing to find treatments for patients with brain disorders. The BBB restricts the uptake of many valuable hydrophilic drugs and limits their efficacy because of the presence of tight junctions, a high metabolic capacity, low pinocytic vesicular traffic, and efficient efflux mechanisms.

Aim

The present study aimed to characterize lactyl stearate-coupled liposomes and their potential for the brain targeting of rifampin (rifampicin).

Method

A liposomal delivery system was prepared for achieving the brain-targeted delivery of rifampin in 21 albino rats utilizing the monocarboxylic acid transport system. Liposomes were prepared by the cast-film method using phosphatidylcholine and cholesterol. Similarly, lactyl stearate-coupled liposomal systems were prepared by casting lactyl stearate film with lipids. These liposomal formulations were characterized for entrapment efficiency, vesicle size, in vitro drug release (using dialysis membrane), and in vivo drug accumulation in various tissues.

Results

Coupling of lactyl stearate to liposomes had a profound influence on entrapment efficiency. Entrapment efficiency was reduced from 41.28 ± 2.02% in uncoupled liposomes to 34.23 ± 1.60% in coupled liposomes. The vesicle size was increased after coupling with lactyl stearate. The in vitro drug release for the uncoupled formulation LIPO-3 was 62.9 ± 3.01% after 24 hours, whereas that of the coupled formulation LIPO-3-Ls-III was 44.5 ± 2.09%. The percentage of rifampin dose recovered from the brain following administration of lactyl stearate-coupled liposomes to albino rats at different time intervals was about 6–8 times higher than with uncoupled liposomes and about 10–12 times higher than with the plain drug solution.

Conclusion

Lactyl stearate-coupled liposomes were better localized within the brain compared to uncoupled liposomes. Lactyl stearate-coupled liposomes could be an excellent carrier system for brain targeting of the hydrophilic drug rifampin.     

Is an Alternative Drug Delivery System Needed for Docetaxel? The Role of Controlling Epimerization in Formulations and Beyond

Purpose

The presence of 7-epidocetaxel in docetaxel injection and in vivo epimerisation has been reported to be the cause for development of tumor resistance to chemotherapy including docetaxel by inducing tumor cell protein cytochrome P450 1B1. The objective of this study was to determine systemic toxicity of Taxotere® containing 10% 7-epidocetaxel and to develop PEGylated liposomal injection that could resist epimerization in vivo. Another need for PEGylated liposomal delivery of docetaxel is to avoid reported hypersensitivity reactions of marketed products like Taxotere® and Duopafei® containing high concentration of tween-80.

Methods

The PEGylated liposomes loaded with docetaxel were prepared using thin film hydration method. The in vivo toxicity of Taxotere® containing 10% 7-epimer was studied in B16F10 experimental metastasis model.

Results

B16F10 experimental metastasis model using C57BL/6 mice injected with Taxotere® containing 10% 7-epimer showed higher weight loss as compared to Taxotere® containing no epimer at single dose of 40 mg/kg indicating higher systemic toxicity. Incubation of PEGylated liposomes with phosphate buffer saline (pH 7.4) containing 0.1% w/v Tween-80 for 48 h showed better resistance to docetaxel degradation when compared with Taxotere® injection indicating better in vivo stability of liposomal docetaxel. In addition, PEGylated liposomes showed enhanced in vitro cytotoxicity, against A549 and B16F10 cells, than Taxotere®.

Conclusion

We can therefore expect less in vivo conversion of liposomal loaded docetaxel into 7-epimer, more passive targeting to tumor tissues, decreased 7-epimer induced systemic toxicity and tumor resistance to chemotherapy compared to Taxotere®. Further in vivo studies are needed to ascertain these facts.     

Targeting of Liposomes via PSGL1 for Enhanced Tumor Accumulation

Purpose

To improve the delivery of liposomes to tumors using P-selectin glycoprotein ligand 1 (PSGL1) mediated binding to selectin molecules, which are upregulated on tumorassociated endothelium.

Methods

PSGL1 was orientated and presented on the surface of liposomes to achieve optimal selectin binding using a novel streptavidin-protein G linker molecule. Loading of PSGL1 liposomes with luciferin allowed their binding to e-selectin and activated HUVEC to be quantified in vitro and their stability, pharmacokinetics and tumor accumulation to be tested in vivo using murine models.

Results

PSGL1 liposomes showed 5-fold (p?<?0.05) greater selectin binding than identically formulated control liposomes modified with ligand that did not contain the selectin binding domain. When added to HUVEC, PSGL1 liposomes showed >7-fold (p?<?0.001) greater attachment than control liposomes. In in vivo studies PSGL1 liposomes showed similar stability and circulation to control liposomes but demonstrated a >3-fold enhancement in the level of delivery to tumors (p?<?0.05).

Conclusions

The technologies and strategies described here may contribute to clinical improvements in the selectivity and efficacy of liposomal drug delivery agents.     

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.     

Optimization of Acoustic Liposomes for Improved In Vitro and In Vivo Stability

Purpose

Liposomes encapsulating perfluoropropane gas, termed acoustic liposomes (ALs), which can serve both for ultrasound (US) imaging and US-mediated gene delivery, have been reported. However, the echogenicity of ALs decreases within minutes in vivo due to gas diffusion and leakage, hindering time-consuming procedures such as contrast-enhanced 3D US imaging and raising the need for improvement of their stability.

Methods

The stability of ALs preparations incorporating increasing ratios of anionic / unsaturated phospholipids, polyethylene glycol (PEG)ylated phospholipid and cholesterol was investigated by measurement of their reflectivity over time using a high-frequency US imaging system, both in vitro and in vivo.

Results

The retention of echogenicity of ALs in vitro is enhanced with increasing molar ratios of PEGylated lipids. Addition of 10 molar percent of an anionic phospholipid resulted in a 31% longer half-life, while cholesterol had the opposite effect. Assessment of the stability of an optimized composition showed a more than 2-fold increase of the detection half-life in mice.

Conclusions

Presence of a PEG coating not only serves to provide ??stealth?? properties in vivo, but also contributes to the retention of the encapsulated gas. The optimized ALs reported here can be used as a contrast agent for lengthier imaging procedures.     

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.     

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.     

Treatment of Experimental Brain Metastasis with MTO-Liposomes: Impact of Fluidity and LRP-Targeting on the Therapeutic Result

Purpose

To test targeted liposomes in an effort to improve drug transport across cellular barriers into the brain.

Methods

Therefore we prepared Mitoxantrone (MTO) entrapping, rigid and fluid liposomes, equipped with a 19-mer angiopeptide as ligand for LDL lipoprotein receptor related protein (LRP) targeting.

Results

Fluid, ligand bearing liposomes showed in vitro the highest cellular uptake and transcytosis and were significantly better than the corresponding ligand-free liposomes and rigid, ligand-bearing vesicles. Treatment of mice, transplanted with human breast cancer cells subcutaneously and into the brain, with fluid membrane liposomes resulted in a significant reduction in the tumor volume by more than 80% and in a clear reduction in drug toxicity. The improvement was mainly depended on liposome fluidity while the targeting contributed only to a minor degree. Pharmacokinetic parameters were also improved for liposomal MTO formulations in comparison to the free drug. So the area under the curve was increased and t_1/2 was extended for liposomes.

Conclusion

Our data show that it is possible to significantly improve the therapy of brain metastases if MTO-encapsulating, fluid membrane liposomes are used instead of free MTO. This effect could be further enhanced by fluid, ligand bearing liposomes.     

In Vitro and In Vivo Biological Evaluation of O-Carboxymethyl Chitosan Encapsulated Metformin Nanoparticles for Pancreatic Cancer Therapy

Purpose

In vitro anticancer effect and in vivo biodistribution and biocompatibility of metformin encapsulated O-Carboxymethyl chitosan nanoparticles were evaluated for its application as pancreatic cancer therapy.

Methods

In vitro studies such as cell migration assay, clonogenic assay, cell cycle analysis and qRT-PCR analysis were done in pancreatic cancer cells (MiaPaCa-2) treated with O-CMC-metformin NPs for evaluating its anticancer potential. In vivo biodistribution studies were carried out by NIR imaging of O-CMC-metformin NPs after tagging it with ICG. In vivo biocompatibility of the NPs was assessed by histopathology analysis of organs from mice administered with the NPs.

Results

In vitro cell migration assay showed marginal effect of NPs on migration property of pancreatic cancer cells (MiaPaCa-2). In vitro clonogenic assay established that the O-CMC-metformin NPs reduced colony formation ability of the cancer cells. While cell cycle analysis showed that the O-CMC-metformin NPs had only minor effect on progression of cell cycle in the cancer cells. qRT-PCR analysis exhibited reduced mRNA expression of p21, vanin 1 and MMP9 in pancreatic cancer cells treated with the nanoparticles. In vivo NIR imaging study showed normal biodistribution pattern of the intravenously injected O-CMC-metformin NPs suggesting normal clearance rate of nanoparticles and no adverse toxicity to the organs.

Conclusions

The biocompatible O-CMC-metformin NPs with anticancer potential and capability for normal biodistribution can be beneficial for the treatment of pancreatic cancer.     

Liposomal Co-Delivery of Omacetaxine Mepesuccinate and Doxorubicin for Synergistic Potentiation of Antitumor Activity

Purpose

Anticancer chemotherapy usually involves the administration of several anticancer drugs that differ in their action mechanisms. Here, we aimed to test whether the combination of omacetaxine mepesuccinate (OMT) and doxorubicin (DOX) could show synergism, and whether the liposomal co-delivery of these two drugs could enhance their antitumor effects in cervical carcinoma model.

Method

OMT-loaded liposomes (OL) were prepared by loading the drug in the lipid bilayers. OL were then electrostatically complexed with DOX, yielding double-loaded liposomes (DOL). DOX-loaded liposomes (DL) were formulated by electrostatic interaction with negatively charged empty liposomes (EL). The combination index (CI) values were calculated to evaluate the synergism of two drugs. In vitro antitumor effects against HeLa cells were measured using CCK-8, calcein staining, and crystal violet staining. In vivo antitumor effects of various liposomes were tested using HeLa cell-bearing mice.

Results

Combination of DOX and OMT had ratio-dependent synergistic activities, with very strong synergism observed at a molar ratio of 4:1 (DOX:OMT). The sizes of EL, DL, OL, and DOL did not significantly differ, but the zeta potentials of DL and DOL were slightly higher than those of OL and EL. In vitro, DOL showed higher antitumor activity than OL, DL or EL in cervical carcinoma HeLa cells. In vivo, unlike other liposomes, DOL reduced the tumor growths by 98.6% and 97.3% relative to the untreated control on day 15 and 25 after the cessation of treatment, respectively.

Conclusions

These results suggest that liposomal co-delivery of DOX and OMT could synergistically potentiate antitumor effects.     

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.     

RAFTsomes Containing Epitope-MHC-II Complexes Mediated CD4+ T Cell Activation and Antigen-Specific Immune Responses

Purpose

To develop a liposome formulation incorporating antigen-presenting cells (APCs) membrane microdomains with enriched epitope/MHC complexes to evaluate the activities of these liposomes (RAFTsomes) to activate T cells and prime immune responses.

Methods

We isolated membrane microdomain structures that contained the epitope/MHC complexes from ovalbumin (OVA) primed dendritic cells (DCs), and reconstituted them on liposomes surface by detergent dialysis. The resulted RAFTsomes were purified by density gradient centrifugation. Their T cell activation functions were evaluated by IL-2 secreting and proliferation assays in vitro. In vivo immune responses and the protective effect against OVA expressing EG.7 tumor challenge were also examined.

Results

Membrane microdomains containing enriched epitope/MHC complexes can be reconstituted into liposomes with defined size and composition. The integrity and activities of these complexes after reconstitution were confirmed by in vitro T cell assays. OVA epitope loaded RAFTsomes injected in vivo resulted in high anti-OVA IgG production (predominantly IgG1). The immunized mice were protected from EG.7 tumor cell inoculation challenge.

Conclusions

Based on these findings, we propose that RAFTsomes can be prepared with unique properties that may be used as an antigen delivery system for immunotherapeutic applications.     

Up-regulating Blood Brain Barrier Permeability of Nanoparticles via Multivalent Effect

Purpose

To investigate the multivalent effect for up-regulating the intracerebral delivery of nanoparticles via receptor-mediated transcytosis.

Methods

Nanoparticles labeled with near-infrared (NIR) fluorophore and different numbers of angiopep-2 peptides that specifically target low-density lipoprotein receptor-related protein (LRP) on the brain capillary endothelial cells were developed. Bio-distribution studies quantified the intracerebral uptakes of these nanoparticles at 2 and 24 h after intravenous injection. In vivo NIR fluorescence imaging, ex vivo autoradiographic imaging and 3D reconstructed NIR fluorescence imaging revealed the nanoparticle distribution pattern in brain. Fluorescence microscopic imaging identified the nanoparticle locations at the cellular level.

Results

The multimetirc association between the angiopep-2 peptides labeled on the nanoparticle and the LRP receptors on the brain capillary endothelial cells significantly increased the intracerebral uptake of the nanoparticles. Nanoparticle Den-Angio4 labeled four angiopep-2 peptides achieved the highest BBB traverse efficacy. After penetrating the BBB, Den-Angio4 distributed heterogeneously and mainly located at hippocampus, striatum and cerebellum in the brains.

Conclusions

The multivalent effect significantly enhances the BBB permeability of nanoparticles. Den-Angio4 as a nanoparticle prototype provides a two order targeted strategy for diagnosis or treatment of central nerver system diseases by first traversing the BBB via receptor-mediated endocytosis and secondly targeting the leisions with high receptor expression level.     

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.     

Protein Antigen Adsorption to the DDA/TDB Liposomal Adjuvant: Effect on Protein Structure, Stability, and Liposome Physicochemical Characteristics

Purpose

Understanding the nature of adjuvant-antigen interactions is important for the future design of efficient and safe subunit vaccines, but remains an analytical challenge. We studied the interactions between three model protein antigens and the clinically tested cationic liposomal adjuvant composed of dimethyldioctadecylammonium (DDA) and trehalose 6,6??-dibehenate (TDB).

Methods

The effect of surface adsorption to DDA/TDB liposomes on colloidal stability and protein physical stability/secondary structure was investigated by dynamic light scattering, circular dichroism, Fourier transform infrared spectroscopy and differential scanning calorimetry.

Results

Bovine serum albumin and ovalbumin showed strong liposome adsorption, whereas lysozyme did not adsorb. Upon adsorption, bovine serum albumin and ovalbumin reduced the phase transition temperature and narrowed the gel-to-liquid phase transition of the liposomes implying interactions with the lipid bilayer. The protein-to-lipid ratio influenced the liposome colloidal stability to a great extent, resulting in liposome aggregation at intermediate ratios. However, no structural alterations of the model proteins were detected.

Conclusions

The antigen-to-lipid ratio is highly decisive for the aggregation behavior of DDA/TDB liposomes and should be taken into account, since it may have an impact on general vaccine stability and influence the choice of analytical approach for studying this system, also/especially at clinically relevant protein-to-lipid ratios.

Microfluidic Preparation of Liposomes to Determine Particle Size Influence on Cellular Uptake Mechanisms

Purpose

This study investigates the cellular uptake and trafficking of liposomes in Caco-2 cells, using vesicles with distinct average diameters ranging from 40.6 nm to 276.6 nm. Liposomes were prepared by microfluidic hydrodynamic flow focusing, producing nearly-monodisperse populations and enabling size-dependent uptake to be effectively evaluated.

Methods

Populations of PEG-conjugated liposomes of various distinct sizes were prepared in a disposable microfluidic device using a simple continuous-flow microfluidic technique. Liposome cellular uptake was investigated using flow cytometry and confocal microscopy.

Results

Liposome uptake by Caco-2 cells was observed to be strongly size-dependent for liposomes with mean diameters ranging from 40.6 nm to 276.6 nm. When testing these liposomes against endocytosis inhibitors, cellular uptake of the largest (97.8 nm and 162.1 nm in diameter) liposomes were predominantly subjected to clathrin-dependent uptake mechanisms, the medium-sized (72.3 nm in diameter) liposomes seemed to be influenced by all investigated pathways and the smallest liposomes (40.6 nm in diameter) primarily followed a dynamin-dependent pathway. In addition, the 40.6 nm, 72.3 nm, and 162.1 nm diameter liposomes showed slightly decreased accumulation within endosomes after 1 h compared to liposomes which were 97.8 nm in diameter. Conversely, liposome co-localization with lysosomes was consistent for liposomes ranging from 40.6 nm to 97.8 nm in diameter.

Conclusions

The continuous-flow synthesis of nearly-monodisperse populations of liposomes of distinct size via a microfluidic hydrodynamic flow focusing technique enabled unique in vitro studies in which specific effects of particle size on cellular uptake were elucidated. The results of this study highlight the significant influence of liposome size on cellular uptake mechanisms and may be further exploited for increasing specificity, improving efficacy, and reducing toxicity of liposomal drug delivery systems.