A detachable coating of cholesterol-anchored PEG improves tumor targeting of cell-penetrating peptide-modified liposomes

Cell-penetrating peptides (CPPs) have been widely used to enhance the membrane translocation of various carriers for many years, but the non-specificity of CPPs seriously limits their utility in vivo. In this study, cholesterol-anchored, reduction-sensitive PEG (first synthesized by our laboratory) was applied to develop a co-modified liposome with improved tumor targeting. Following optimization of the formulation, the in vitro and in vivo properties of the co-modified liposome were evaluated. The co-modified liposome had a much lower cellular uptake and tumor spheroid uptake, but a much higher tumor accumulation compared to CPP-modified liposome, indicating the non-specific penetration of CPPs could be attenuated by the outer PEG coating. With the addition of exogenous reducing agent, both the in vitro and in vivo cellular uptake was markedly increased, demonstrating that the reduction-sensitive PEG coating achieved a controllable detachment from the surface of liposomes and did not affect the penetrating abilities of CPPs. The present results demonstrate that the combination of cholestervsitive PEG and CPPs is an ideal alternative for the application of CPP-modified carriers in vivo.KEY WORDS: Cell penetrating peptide, Reduction-sensitive PEG, Tumor targeting, Cholesterol, Liposome     

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.     

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.     

Synergistic targeted delivery of payload into tumor cells by dual-ligand liposomes co-modified with cholesterol anchored transferrin and TAT

This study was mainly focused on developing a dual-ligand liposomal delivery system to enhance both targeting specificity and cellular uptake. The specific ligand transferrin (TF) and the cationic cell-penetrating peptide TAT were connected with cholesterol via a polyethylene glycol (PEG) spacer to prepare the dual-ligand liposomes (TAT/TF-PEG-LP). Then the in vitro cellular uptake by three kinds of cells that possessed different expressing levels of transferrin receptor (TFR) and the in vivo delivery efficiency were evaluated. Compared to the single-ligand TAT or TF modified liposomes (TAT-PEG-LP or TF-PEG-LP), TAT/TF-PEG-LP exhibited the enhanced cellular uptake and selectivity via the synergistic effect of both ligands in vitro. The ex vivo fluorescence imaging of tumors, the qualitative observation of tumor frozen section and the quantitative determination of cellular uptake in tumor tissues altogether showed the in vivo delivery efficiency of TAT/TF-PEG-LP was higher than that of other liposomes. In conclusion, the dual-ligand liposomes co-modified with TF and TAT possessed a strong capability for synergistic targeted delivery of payload into tumor cells both in vitro and in vivo.     

PCM and TAT co-modified liposome with improved myocardium delivery: in vitro and in vivo evaluations

In this study, PCM and TAT co-modified liposome was developed as a novel drug carrier for myocardium delivery with evaluation of its in vitro and in vivo properties. Liposomes containing fluorescent probe coumarin-6 were prepared by thin-film hydration. The PCM ligands specifically bind to the PCM receptors in the extracellular connective tissue of primary myocardium cells (MCs), while the TAT ligands functioned as a classical cell penetrating peptide to make liposomes internalized by MCs. The unmodified liposome (L), PCM-modified liposome (PL), TAT-modified liposome (TL) and PCM and TAT co-modified liposome (PTL) were prepared and characterized. The cellular uptake and intracellular distribution of various liposomes by MCs demonstrated that PTL had the best delivery capability. Peptide inhibition assay indicated that the uptake of PL could be inhibited by PCM. However, TAT could almost not suppress the uptake of TL. In addition, the CCK-8 experiments showed that liposomes had low cytotoxicity. In vivo fluorescent images of frozen sections and HPLC-fluorescence analysis further demonstrated that PTL had highest myocardium distribution. The results of this study demonstrated that PCM and TAT co-modifying could improve the myocardial targeting ability of liposome.     

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.     

Polydopamine-Based Surface Modification for the Development of Peritumorally Activatable Nanoparticles

Purpose

To create poly(lactic-co-glycolic acid) (PLGA) nanoparticles (NPs), where a drug-encapsulating NP core is covered with polyethylene glycol (PEG) in a normal condition but exposes a cell-interactive TAT-modified surface in an environment rich in matrix metalloproteinases (MMPs).

Methods

PLGA NPs were modified with TAT peptide (PLGA-pDA-TAT NPs) or dual-modified with TAT peptide and a conjugate of PEG and MMP-substrate peptide (peritumorally activatable NPs, PANPs) via dopamine polymerization. Cellular uptake of fluorescently labeled NPs was observed with or without a pre-treatment of MMP-2 by confocal microscopy and flow cytometry. NPs loaded with paclitaxel (PTX) were tested against SKOV-3 ovarian cancer cells to evaluate the contribution of surface modification to cellular delivery of PTX.

Results

While the size and morphology did not significantly change due to the modification, NPs modified with dopamine polymerization were recognized by their dark color. TAT-containing NPs (PLGA-pDA-TAT NPs and PANPs) showed changes in surface charge, indicative of effective conjugation of TAT peptide on the surface. PLGA-pDA-TAT NPs and MMP-2-pre-treated PANPs showed relatively good cellular uptake compared to PLGA NPs, MMP-2-non-treated PANPs, and NPs with non-cleavable PEG. After 3 h treatment with cells, PTX loaded in cell-interactive NPs showed greater toxicity than non-interactive ones as the former could enter cells during the incubation period. However, due to the initial burst drug release, the difference was not as clear as microscopic observation.

Conclusions

PEGylated polymeric NPs that could expose cell-interactive surface in response to MMP-2 were successfully created by dual modification of PLGA NPs using dopamine polymerization.     

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.     

Asymmetric Peptide Dendrimers are Effective Linkers for Antibody-Mediated Delivery of Diverse Payloads to B Cells in Vitro and in Vivo

Purpose

Safe, targeted delivery of therapeutics remains a focus of drug/gene delivery, the aim being to achieve optimal efficacy while minimising off-target delivery. Dendrimers have a vast array of potential applications and have great potential as gene and drug delivery tools. We previously reported the development of peptide dendrimers that effectively complexed DNA and that have distinct advantages over conventional spherical dendrimers. Here, to expand the application of peptide-based low generation dendrimers we tested their capacity to be transformed into linkers for antibody-based targeting of diverse payloads.

Methods

Peptide-based low-generation asymmetric dendrimers were generated and conjugated to partially-reduced antibodies specific for B cell surface antigens or an irrelevant antigen. Preservation of antigen binding by the antibodies and targeting of the conjugated dendrimers carrying a small molecule (biotin) or plasmid DNA payloads was tested.

Results

Peptide-based low generation dendrimers were efficiently and site-specifically conjugated to antibodies with retention of antigen-binding capacity. Altering the branching termini of dendrimers facilitated delivery of diverse payloads in vitro and in vivo.

Conclusions

We propose that safe, non-toxic peptide dendrimers, which are readily synthesised and modifiable for a variety of applications, form the basis of a new family of biocompatible “linkers” with substantial potential for targeted delivery applications.     

Multi-Compartmental Vaccine Delivery System for Enhanced Immune Response to gp100 Peptide Antigen in Melanoma Immunotherapy

Purpose

To develop a multi-compartmental vaccine delivery system for safe and efficient delivery of the gp100 peptide antigen in melanoma immunotherapy.

Methods

Water-in-oil-in-water (W/O/W) multiple emulsion-based multi-compartmental vaccine delivery system containing the gp100 peptide was prepared by a two-step emulsification method. In vivo prophylactic and active immunization effectiveness of the novel squalane oil-containing gp100 vaccine was evaluated in the murine B16 melanoma model and compared with that of an incomplete Freund’s adjuvant (IFA)-based vaccine.

Results

Morphological evaluation of the W/O/W multiple emulsions showed that the oil-droplets were homogenously dispersed with the gp100 peptide encapsulated in an inner aqueous phase. Immunization with the gp100 peptide delivered in the W/O/W multiple emulsions-based vaccine resulted in increased protection against tumor challenge compared to IFA-based vaccine (p?<?0.05, n?=?8) signifying induction of enhanced anti-tumor immunity. In addition, serum Th1 cytokine levels and immuno-histochemistry of excised tumor tissues indicated activation and local infiltration of antigen specific cytotoxic T-lymphocytes into and/or surrounding the tumor mass. Moreover, the newly developed vaccine formulation did not induce any overt systemic toxicity.

Conclusion

Novel W/O/W multiple emulsions-based vaccine efficiently delivers the gp100 peptide antigen to induce cell-mediated anti-tumor immunity and offers an alternate, safe vaccine delivery system.     

Novel Comb-Shaped PEG Modification Enhances the Osteoclastic Inhibitory Effect and Bone Delivery of Osteoprotegerin After Intravenous Administration in Ovariectomized Rats

Purpose

Recombinant osteoprotegerin (OPG) has been proven to be useful for treating various bone disorders such as osteoporosis. To improve its in vivo pharmacological effect, OPG was conjugated to novel comb-shaped co-polymers of polyethylene glycol (PEG) allylmethylether and maleamic acid (poly(PEG), 5?kDa). Biodistribution and bioactivity were evaluated.

Methods

OPG was conjugated via lysine to poly(PEG) and to linear PEG (0.5?kDa and 5?kDa). Poly(PEG)-OPG was compared with linear PEG0.5k-OPG and PEG5k-OPG in terms of in vitro and in vivo efficacy and bone distribution.

Results

The in vitro receptor binding study showed that poly(PEG)-OPG could be the most bioactive among the three PEG-OPG derivatives. Pharmacokinetic studies in ovariectomized (OVX) rats showed that serum half-life and AUC of poly(PEG)-OPG were comparable with those of linear PEG-OPG derivatives. For in vivo pharmacological effect, poly(PEG)-OPG showed the strongest inhibitory effect on bone resorption activity in OVX rats. Poly(PEG)-OPG demonstrated enhanced bone marrow distribution with higher selectivity than linear PEG5k-OPG.

Conclusion

Poly(PEG) modification could provide longer residence time in serum and higher bone-marrow specific delivery of OPG, leading to a higher in vivo pharmacological effect.     

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.     

Ultrasound-enhanced chemotherapy of drug-resistant breast cancer tumors by micellar-encapsulated Paclitaxel

Background and aim

Currently, delivery of the poorly water-soluble chemotherapeutic agent paclitaxel is associated with a substantial array of systemic toxicities and results in low-efficiency tumor treatment. A novel on-demand delivery system based on paclitaxel encapsulated in polymeric micelles in conjunction with triggered release of the drug by local ultrasonic irradiation of the tumor was evaluated in vitro and in vivo using a drug-resistant MCF7/ADmt breast cancer human cell line.

Method

The effects of local ultrasonic tumor irradiation on cellular proliferation and intracellular drug uptake were compared for a developmental micellar paclitaxel formulation (SYP-PM) and a currently available clinical intravenous formulation of paclitaxel.

Results

Without ultrasound, the uptake of paclitaxel from the micellar formulation was significantly lower than that from the clinical formulation, which is advantageous for preventing unwanted drug interactions with healthy tissues in vivo. When micellar encapsulation was combined with ultrasonically triggered release, drug uptake from micellar paclitaxel was increased more than 20-fold and cellular proliferation was inhibited by nearly 90%. Without ultrasound, the clinical formulation of paclitaxel and SYP-PM manifested low efficacy in vivo, whereas injections of SYP-PM combined with ultrasound resulted in complete tumor resolution.

Conclusion

The ability of micellar-encapsulated paclitaxel to exert a significant cytotoxic effect only when subjected to ultrasound proves promising for the development of a tumor-targeted ultrasound-enhanced paclitaxel delivery system for clinical application. This treatment modality could be successfully used for the therapy of both drug-sensitive and drug-resistant tumors. The major advantages of a micellar formulation of paclitaxel combined with local tumor sonication are the aqueous base of the drug formulation, reduced systemic toxicity, potential for tumor targeting, and on-demand delivery of drug to tumor cells.     

Topical Anti-Inflammatory Potential of Quercetin in Lipid-Based Nanosystems: In Vivo and In Vitro Evaluation

Purpose

To develop quercetin-loaded phospholipid vesicles, namely liposomes and PEVs (Penetration Enhancer-containing Vesicles), and to investigate their efficacy on TPA-induced skin inflammation.

Methods

Vesicles were made from a mixture of phospholipids, quercetin and polyethylene glycol 400 (PEG), specifically added to increase drug solubility and penetration through the skin. Vesicle morphology and self-assembly were probed by Cryo-Transmission Electron Microscopy and Small/Wide Angle X-ray Scattering, as well as the main physico-chemical features by Light Scattering. The anti-inflammatory efficacy of quercetin nanovesicles was assessed in vivo on TPA-treated mice dorsal skin by the determination of two biomarkers: oedema formation and myeloperoxidase activity. The uptake of vesicles by 3T3 fibroblasts was also evaluated.

Results

Small spherical vesicles were produced. Their size and lamellarity was strongly influenced by the PEG content (0%, 5%, 10% v/v). The administration of vesicular quercetin on TPA-inflamed skin resulted in an amelioration of the tissue damage, with a noticeable attenuation of oedema and leukocyte infiltration, especially using 5% PEG-PEVs, as also confirmed by confocal microscopy. In vitro studies disclosed a massive uptake and diffusion of PEVs in dermal fibroblasts.

Conclusions

The proposed approach based on quercetin vesicular formulations may be of value in the treatment of inflammatory skin disorders.     

Trimethyl Chitosan-Cysteine Nanoparticles for Systemic Delivery of TNF-α siRNA via Oral and Intraperitoneal Routes

Purpose

The lack of effective delivery vehicles impedes in vivo applications of siRNA. The trimethyl chitosan-cysteine (TC) nanoparticles (NPs) were developed for in vivo delivery of tumor necrosis factor α (TNF-α) siRNA via oral gavage and intraperitoneal injection.

Methods

The nanoparticles formulated from TC conjugate of 100, 200, and 500 kDa were prepared through ionic gelation with sodium tripolyphosphate, termed as TC100 NPs, TC200 NPs, and TC500 NPs, respectively. They were evaluated in terms of stability, siRNA protection, cellular uptake and TNF-α knockdown in peritoneal exudates macrophage cells (PECs), and in vivo TNF-α silencing in acute hepatic injury mice.

Results

TC100 NPs exhibited poor stability in simulated physiological environment compared to TC200 NPs and TC500 NPs. Compared to TC500 NPs, TC200 NPs could significantly enhance in vitro and in vivo cellular uptake by PECs and facilitate cytoplasmic siRNA release, resulting in high in vitro and in vivo TNF-α knockdown. Superior TNF-α suppressing level was obtained with TC200 NPs via oral gavage rather than intraperitoneal injection.

Conclusions

The efficacies of in vivo TNF-α silencing were related to the molecular weight of TC conjugate and the administration route, which would assist in the rational design of siRNA vehicles.     

Brain Delivery of NAP with PEG-PLGA Nanoparticles Modified with Phage Display Peptides

Purpose

A phage-displayed peptide TGN was used as a targeting motif to help the delivery of NAP-loaded nanoparticles across the blood–brain barrier (BBB), which sets an obstacle for brain delivery of NAP in vivo.

Methods

Intracerebroventricular injection of Aβ_1-40 into mice was used to construct in vivo model of Alzheimer’s disease. The water maze task was performed to evaluate the effects of the NAP formulations on learning and memory deficits in mice. The neuroprotective effect was tested by detecting acetylcholinesterase (AChE) and choline acetyltransferase (ChAT) activity and conducting histological assays.

Results

Intravenous administration of NAP-loaded TGN modified nanoparticles (TGN-NP/NAP) has shown better improvement in spatial learning than NAP solution and NAP-loaded nanoparticles in Morris water maze experiment. The crossing number of the mice with memory deficits recovered after treatment with TGN-NP/NAP in a dose dependent manner. Similar results were also observed in AChE and ChAT activity. No morphological damage and no detectable Aβ plaques were found in mice hippocampus and cortex treated with TGN-NP/NAP.

Conclusions

TGN modified nanoparticles could be a promising drug delivery system for peptide and protein drug such as NAP to enter the brain and play the therapeutic role.     

NIR-/pH-Responsive Drug Delivery of Functionalized Single-Walled Carbon Nanotubes for Potential Application in Cancer Chemo-Photothermal Therapy

Purpose

To establish a NIR (near infrared)-/pH-responsive and sustained-release tumor-targeting drug delivery system (SWNT-PEI/DOX/NGR).

Methods

Functionalized SWNTs with polymerised polymeric poly(ethylene imine) was linked NGR (Asn-Gly-Arg) tumor-targeting peptide by DSPE-PEG2000-Maleimide via the maleimide group and sulfhydryl group of cysteine, in the end, doxorubicin (DOX) was attached to SWNT-PEI to obtain a SWNT-PEI/DOX/NGR delivery system.

Results

The SWNT-PEI/DOX/NGR delivery system has significantly sustained-release effect and the slow release of DOX in normal tissues contribute to reduced systemic toxicity, while under 808 nm NIR laser irradiation or under lower pH environment the release of DOX can be accelerated.

Conclusions

Due to hyperthermia sensitizer effect of DOX, chemo-photothermal exemplified by SWNT-PEI/DOX/NGR tumor-targeting delivery system is a promising approach to anticancer therapy in vivo or in vitro.     

Improving Intracellular Doxorubicin Delivery Through Nanoliposomes Equipped with Selective Tumor Cell Membrane Permeabilizing Short-Chain Sphingolipids

Purpose

To improve nanoliposomal-doxorubicin (DoxNL) delivery in tumor cells using liposome membrane-incorporated short-chain sphingolipids (SCS) with selective membrane-permeabilizing properties. DoxNL bilayers contained synthetic short-chain derivatives of known membrane microdomain-forming sphingolipids; C₈-glucosylceramide (C₈-GluCer), C₈-galactosylceramide (C₈-GalCer) or C₈-lactosylceramide (C₈-LacCer).

Methods

DoxNL enriched with C₈-GluCer or C₈-GalCer were developed, optimized and characterized with regard to size, stability and drug retention. In vitro cytotoxic activity was studied in a panel of human tumor cell lines and normal cells. Intracellular Dox delivery was measured by flow cytometry and visualized by fluorescence microscopy. For a further understanding of the involved drug delivery mechanism confocal microscopy studies addressed the cellular fate of the nanoliposomes, the SCS and Dox in living cells.

Results

C₈-LacCer-DoxNL aggregated upon Dox loading. In tumor cell lines SCS-DoxNL with C₈-GluCer or C₈-GalCer demonstrated strongly increased Dox delivery and cytotoxicity compared to standard DoxNL. Surprisingly, this effect was much less pronounced in normal cells. Nanoliposomes were not internalized, SCS however transfered from the nanoliposomal bilayer to the cell membrane and preceded cellular uptake and subsequent nuclear localization of Dox.

Conclusion

C₈-GluCer or C₈-GalCer incorporated in DoxNL selectively improved intracellular drug delivery upon transfer to tumor cell membranes by local enhancement of cell membrane permeability.     

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.     

Fatty Acid-Based Strategy for Efficient Brain Targeted Gene Delivery

Purpose

To investigate a fatty acid-based strategy for efficient brain targeted gene delivery and to understand mechanism(s) of this small molecule-mediated brain gene delivery strategy.

Methods

A series of fatty acids (FAs) were conjugated with polyethylenimine (PEI_25k). A near-infrared fluorescence probe, IR820, was used to study in vivo and ex vivo brain targeting ability of these fatty acid-PEI conjugates (FA-PEIs). Brain uptake of FA-PEI_25k/rhodamine-6-isothiocyanate (RITC)-labeled DNA nanoparticles was investigated via a fluorescence imaging method. Moreover, pEGFP was used as a model gene to study in vitro and in vivo transfection effect of the ideal FA-PEI_25k conjugate.

Results

FA modification did not have interference with the complexation between DNA and the PEI_25k. The FA-PEI_25k conjugates showed excellent brain targeting ability compared with unmodified PEI_25k. Among these FA-PEI_25k conjugates studied, myristic acid (MC)-PEI_25k showed sustained brain distribution profile and higher brain DNA uptake. Furthermore, MC-PEI_25k/pEGFP nanoparticles was able to achieve efficient in vitro and in vivo gene transfection. GFP expression was observed at different brain regions in vivo.

Conclusions

These results demonstrated that the small molecule fatty acid, particularly myristic acid-based brain gene delivery strategy, is promising to mediate efficient gene transfection in the brain.