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.     

Spatiotemporally Controlled Co-delivery of Anti-vasculature Agent and Cytotoxic Drug by Octreotide-Modified Stealth Liposomes

Purpose

Both combretastatin A-4 (CA-4) and doxorubicin (DOX) was loaded in different form in a targeted nanomedicine in order to achieve the active delivery of these two drugs followed by sequentially suppressing tumor vasculature and tumor cells.

Methods

Octreotide-modified stealth liposomes loaded with CA-4 and DOX (Oct-L[CD]) were prepared and characterized. Then in vitro release, cellular uptake, in vitro antitumor effect, pharmacokinetics, in vivo sequential killing effect, in vivo antitumor efficacy against somatostatin receptor (SSTR) positive cells, as well as the action mechanism of such system, were studied.

Results

A rapid release of CA-4 followed by a slow release of DOX was observed in vitro. The active targeted liposomes Oct-L[CD] showed a specific cellular uptake through ligand-receptor interaction and a higher antitumor effect in vitro against SSTR-positive cell line. The in vivo sequential killing effect of such system was found as evidenced by the fast inhibition of blood vessels and slow apoptosis-inducing of tumor cells. Oct-L[CD] also exhibited the strongest antitumor effect in MCF-7 subcutaneous xenograft models.

Conclusions

Oct-modified co-delivery system may have great potential as an effective carrier for cancer therapy.     

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.

     

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.     

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.     

Hyaluronic Acid Derivative-Based Self-Assembled Nanoparticles for the Treatment of Melanoma

Purpose

Hyaluronic acid-ceramide (HACE)-based nanoparticles (NPs) were developed for the targeted delivery of doxorubicin (DOX), and their antitumor efficacy for melanoma was evaluated.

Methods

DOX-loaded HACE-based self-assembled NPs were prepared and their physicochemical properties were characterized. The in vitro cytotoxicity of HACE was measured using an MTS-based assay. The cellular uptake efficiency of DOX into mouse melanoma B16F10 cells was assessed by confocal laser scanning microscopy and flow cytometry. Tumor growth and body weight were monitored after the intratumoral and intravenous injection of DOX-loaded NPs into a B16F10 tumor-bearing mouse model.

Results

DOX-loaded NPs, with a mean diameter of ~110?nm, a narrow size distribution, and high drug entrapment efficiency, were prepared. A sustained DOX release pattern was shown, and drug release was enhanced at pH 5.5 compared with pH 7.4. The cytotoxicity of HACE to B16F10 cells was negligible. It was assumed that DOX was taken up into the B16F10 cells through receptor-mediated endocytosis. A significant inhibitory effect was observed on tumor growth, without any serious changes in body weight, after the injection of DOX-loaded NPs into the B16F10 tumor-bearing mouse model.

Conclusions

DOX-loaded HACE-based NPs were successfully developed and their antitumor efficacy against B16F10 tumors was demonstrated.     

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.     

Novel Core-Interlayer-Shell DOX/ZnPc Co-loaded MSNs@ pH-Sensitive CaP@PEGylated Liposome for Enhanced Synergetic Chemo-Photodynamic Therapy

Purpose

This work was intended to develop novel doxorubicin (DOX)/zinc (II) phthalocyanine (ZnPc) co-loaded mesoporous silica (MSNs)@ calcium phosphate (CaP)@PEGylated liposome nanoparticles (NPs) that could efficiently achieve collaborative anticancer therapy by the combination of photodynamic therapy (PDT) and chemotherapy. The interlayer of CaP could be utilized to achieve pH-triggered controllable drug release, promote the cellular uptake, and induce cell apoptosis to further enhance the anticancer effects.

Methods

MSNs were first synthesized as core particles in which the pores were diffusion-filled with DOX, then the cores were coated by CaP followed by the liposome encapsulation with ZnPc to form the final DOX/ZnPc co-loaded MSNs@CaP@PEGylated liposome.

Results

A core-interlayer-shell MSNs@CaP@PEGylated liposomes was developed as a multifunctional theranostic nanoplatform. In vitro experiment indicated that CaP could not only achieve pH-triggered controllable drug release, promote the cellular uptake of the NPs, but also generate high osmotic pressure in the endo/lysosomes to induce cell apoptosis. Besides, the chemotherapy using DOX and PDT effect was achieved by the photosensitizer ZnPc. Furthermore, the MSNs@CaP@PEGylated liposomes showed outstanding tumor-targeting ability by enhanced permeability and retention (EPR) effect.

Conclusions

The novel prepared MSNs@CaP@PEGylated liposomes could serve as a promising multifunctional theranostic nanoplatform in anticancer treatment by synergic chemo-PDT and superior tumor-targeting ability.

     

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.     

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.     

Bioactive Lipids-Based pH Sensitive Micelles for Co-Delivery of Doxorubicin and Ceramide to Overcome Multidrug Resistance in Leukemia

Purpose

Construction of a novel PEGylated bioactive lipids-based micelle system for co-delivery of doxorubicin (DOX) and short chain ceramide (C6-ceramide) to overcome multidrug resistance in leukemia.

Methods

The PEGylated bioactive lipids-based micelle system was constructed via electrostatic and hydrophobic interactions among DOX, bioactive lipids PazPC and C6-ceramide. The micellar formulation was characterized in terms of size, zeta potential, stability and release behavior, etc., and in vitro cytotoxicity, in vivo antitumor efficacy and the underlying mechanism were further evaluated.

Results

This novel micellar system showed small size (~15 nm), high drug encapsulation efficiency (>90%), good stability and endosomal acid-triggered release of DOX. Synergistic cytotoxic effects between DOX and bioactive lipid C6-ceramide in P-gp overexpressing drug resistant leukemia P388/ADR cells were observed. The mechanistic studies demonstrated that modulation of drug efflux system and induction of apoptotic effects by lipids were responsible for the synergistic effects between DOX and C6-ceramide in drug resistant leukemia P388/ADR cells. Using an in-vivo P388/ADR leukemia mouse model, the median survival time of the DOX-loaded PEGylated micelles with PazPC and C6-ceramide as major components was significantly greater than that of free DOX and control group.

Conclusions

We developed a novel pH sensitive bioactive lipids-based micellar formulation which could potentially be useful in delivering chemotherapeutic drug DOX and provide a novel strategy to increase the therapeutic index for drug resistant leukemia treatment.     

Simulation of Stimuli-Responsive and Stoichiometrically Controlled Release Rate of Doxorubicin from Liposomes in Tumor Interstitial Fluid

Purpose

To simulate the stimuli-responsive and stoichiometrically controlled doxorubicin (DOX) release from liposomes in in vivo tumor interstitial fluid (TIF), the effect of ammonia concentration and pH on the DOX release from liposomes in human plasma at 37°C was quantitatively evaluated in vitro and the release rate was calculated as a function of ammonia concentration and pH.

Methods

Human plasma samples spiked with DOX-loaded PEGylated liposomes (PLD) or Doxil^®, containing ammonia (0.3–50 mM) at different pH values, were incubated at 37°C for 24 h. After incubation, the concentration of encapsulated DOX in the samples was determined by validated solid-phase extraction (SPE)-SPE-high performance liquid chromatography.

Results

Accelerated DOX release (%) from liposomes was observed as the increase of ammonia concentration and pH of the matrix, and the decrease of encapsulated DOX concentration. The release rate was expressed as a function of the ammonia concentration and pH by using Henderson-Hasselbalch equation.

Conclusions

The DOX release from PLD in TIF was expressed as a function ammonia concentration and pH at various DOX concentrations. Further, it was found that the DOX release from liposomes in a simulated TIF was more than 15 times higher than in normal plasma.

     

Freeze-Anneal-Thaw Cycling of Unilamellar Liposomes: Effect on Encapsulation Efficiency

Purpose

Freeze-thaw cycling is an important processing step in the preparation of liposomes that leads to the encapsulation of drug molecules. There is considerable variability in the number of freeze-thaw cycles reported in the literature. This work is designed to aid in liposomal formulation design by gaining an insight into the drug encapsulation process and an understanding of liposome stabilization during various thawing conditions.

Methods

The effects of different thawing temperatures, as well as “annealing” at subzero temperatures on a liposome formulation, are reported here.

Results

Two freeze-anneal-thaw (FA_NNT) cycles (freezing to ?196°C, annealing at ?1.4°C for ~30 min, thawing at 65°C) resulted in the maximum predicted encapsulation efficiency without causing any significant change in particle size or zeta potential. Annealing at ?22°C was shown to be destabilizing due to limited hydration of the liposomes in the frozen state.

Conclusions

It was shown that two important processes are occurring during the FA_NNT cycling that affect liposome encapsulation efficiency. The first is drug diffusion in the frozen state and the second is fusion/destabilization of the liposomes. This is the first report on the annealing of liposomes and understanding the mechanism of drug encapsulation using the freeze-thaw cycling method.     

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.     

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.     

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.

Development and Evaluation of Tri-Functional Immunoliposomes for the Treatment of HER2 Positive Breast Cancer

Purpose

Trastuzumab combined with Doxorubicin (DOX) demonstrates significant clinical activity in human epidermal growth factor receptor-2 (HER2)-positive breast cancer (BC). However, emergence of treatment resistance and trastuzumab associated cardiotoxicity remain clinical challenges. In an effort to improve patient outcome, we have developed and evaluated novel tri-functional immunoliposomes (TFIL) that target HER2-receptors on BC cells and CD3-receptors on T-lymphocytes, and deliver DOX.

Methods

Trastuzumab (anti-HER2) and OKT-3 (anti-CD3) antibodies were conjugated to liposomes using a micelle-transfer method. Cytotoxicity of targeted immunoliposomes loaded with DOX was examined in vitro on HER2-positive BC cells (BT474), with peripheral blood monocytic cells (PBMC) as immune effector cells.

Results

TFIL demonstrated high antibody-liposome conjugation ratios (100–130 μg protein/μmol phospholipid) and cargo capacity (0.21 mol:mol drug:lipid), highly efficient DOX loading (>90%), a particle size favorable for extended circulation (~150 nm), and good stability (up to 3 months at 4°C). In the presence of PBMCs, TFIL showed complete killing of BT474 cells, and were superior to mono-targeted trastuzumab-bearing liposomes, non-targeted liposomes, and free Trastuzumab and DOX.

Conclusions

Novel anti-HER2xCD3?+?DOX TFIL show promise as a means to both engage immune cells against HER2 positive breast cancer cells and deliver chemotherapy, and have the potential to improve clinical outcomes.

     

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.     

Protein Encapsulation in Unilamellar Liposomes: High Encapsulation Efficiency and A Novel Technique to Assess Lipid-Protein Interaction

Purpose

To encapsulate a large amount of protein (superoxide dismutase, SOD) into unilamellar liposomes using a simple process and to investigate the lipid-protein interaction.

Method

To achieve protein encapsulation, preformed unilamellar empty liposomes were mixed with SOD and subjected to freeze-thaw cycling. To investigate the lipid-protein interaction, a novel light scattering technique was used.

Results

Up to 50% protein encapsulation was achieved at ??150?nm. There was no significant change in particle size following the freeze-thaw cycling. SOD had a strong interaction with DPPC liposomes containing high concentration of cholesterol. Light scattering data revealed that in some cases the SOD molecules were present inside the lipid bilayer.

Conclusions

The method reported here allows great flexibility in the manufacturing process as the liposome preparation and protein-loading operations can be separated. Accordingly, empty liposomes can be prepared without concern about protein stability, making the manufacturing process more flexible and easy to control and ultimately leading to improved product quality. To explain the SOD-lipid interaction, a ??pocket-embedding?? theory was proposed. The encapsulation method reported here can be applied to hydrophilic small molecules as well as most hydrophilic proteins to achieve high encapsulation efficiency.     

Near-infrared light-activated IR780-loaded liposomes for anti-tumor angiogenesis and Photothermal therapy

Tumor angiogenesis is a key step in the process of tumor development, and antitumor angiogenesis has a profound influence on tumor growth. Herein we report a dual-function drug delivery system comprising a Near-infrared (NIR) dye and an anti-angiogenic drug within liposomes (Lip-IR780-Sunitinib) for enhanced antitumor therapy. The hydrophobic NIR dye IR780 was loaded into the liposome phospholipid bilayer, and the bilayer would be disrupted by laser irradiation so that anti-angiogenic drug sunitinib release would be activated remotely at the tumor site. The released hydrophilic sunitinib could potentially target multiple VEGF receptors on the tumor endothelial cell surface to inhibit angiogenesis. Meanwhile, IR780-loaded liposomes kill the cancer cells by photothermal therapy. Lip-IR780-Sunitinib exhibited enhanced anti-tumor and anti-angiogenic effects in vitro and in vivo. This system facilitates easy and controlled release of cargos to achieve anti-tumor angiogenesis and photothermal therapy.