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.     

Folate and TAT Peptide Co-Modified Liposomes Exhibit Receptor-Dependent Highly Efficient Intracellular Transport of Payload In Vitro and In Vivo

Purpose

Using different chain lengths of PEG as linkers to develop a novel folate (FA) and TAT peptide co-modified doxorubicin (DOX)-loaded liposome (FA/TAT-LP-DOX) and evaluate its potential for tumor targeted intracellular drug delivery.

Methods

FA/TAT-LP-DOX was prepared by pH gradient method and post-insertion method and the optimal ligand density was screened by MTT assay. In vitro evaluation was systematically performed through cytotoxicity assay, cellular uptake studies, subcellular localization and cellular uptake mechanism in folate receptor (FR) over-expressing KB tumor cells. In vivo tumor targeted delivery of FA/TAT-LP-DOX was also studied by in vivo fluorescence imaging in a murine KB xenograft model.

Results

The particle size and zeta potential determination indicated that FA and TAT were successfully inserted into the liposome and cationic TAT peptide was completely shielded. With the optimal ligand density (5% of FA and 2.5% TAT), the FA/TAT-LP-DOX exhibited improved cytotoxity and cellular uptake efficiency compared with its single-ligand counterparts (FA-LP-DOX and PEG/TAT-LP-DOX). Competitive inhibition and uptake mechanism experiments revealed that FA and TAT peptide played a synergistic effect in facilitating intracellular transport of the liposome, and association between FA and FA receptors activated this transport process. In vivo imaging further demonstrated the superiority of FA/TAT-LP in tumor targeting and accumulation.

Conclusions

Folate and TAT peptide co-modified liposome using different chain lengths of PEG as linkers may provide a useful strategy for specific and efficient intracellular 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.

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.     

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.     

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.     

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.     

Controlled Nucleation of Lipid Nanoparticles

Purpose

We describe a nucleation-based method which allows for the generation of monodisperse lipid nanoparticles over a range of diameters. Using a set of novel zwitterionic lipids and inverse phosphocholine lipids with pKas ranging from 2 to 5, we showed how the hydrodynamic diameter of lipid nanoparticles can be systematically manipulated over a 60?nm to 500?nm size range.

Method

Lipid nanoparticles were prepared by adding an anti-solvent, such as water, to the organic phase containing the lipid components. This led to super-saturation and the spontaneous formation of particles.

Results

The growth and final particle size was controlled by the ratio of the components in the ternary system: lipid, organic solvent and aqueous phase. Particles with diameter below 125?nm were formed under conditions where the super-saturation coefficient was between 2.3 and 20. PEG-lipid served as an efficient growth inhibitor except at very high and low lipid concentrations. Encapsulation efficiency of siRNA into lipid nanoparticles was shown to be pH-dependent and requires the protonation of the anionic carboxylate groups of the zwitterionic lipids, emphasizing the importance of electrostatic forces.

Conclusion

This process enables high encapsulation efficiency of nucleic acids and allows the size of lipid nanoparticles to be controlled.     

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.     

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.     

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

Purpose

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

Methods

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

Results

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

Conclusions

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

修饰脂质体的可断裂聚乙二醇脂质衍生物的研究进展

聚乙二醇脂质衍生物可增加脂质体的稳定性，延长其体内循环时间。传统的长循环材料由于连接聚乙二醇与脂质的化学键太稳定，导致脂质体内容物释放延迟而影响药效。近年来提出可断裂聚乙二醇脂质衍生物的概念，该类衍生物具有可以在人的生理或病理条件下断裂的性质，能够延长脂质体体内循环时间，在到达靶部位后由于聚乙二醇已经从脂质体表面脱落，脂质体可以与病变细胞结合，从而将药物送入细胞。本文综述了可断裂聚乙二醇脂质衍生物的种类、断裂类型、在脂质体中的应用概况及在应用中的优势和局限。     

Prevention of Orthopedic Device-Associated Osteomyelitis Using Oxacillin-Containing Biomineral-Binding Liposomes

Purpose

To develop novel biomineral-binding liposomes (BBL) for the prevention of orthopedic implant associated osteomyelitis.

Methods

A biomineral-binding lipid, alendronate-tri(ethyleneglycol)-cholesterol conjugate (ALN-TEG-Chol), was synthesized through Cu(I)-catalyzed Huisgen 1,3-dipolar cycloaddition (a versatile click reaction). Mixing with other excipients, the new lipid was used to develop BBL. Thermodynamic behavior was studied by differential scanning calorimetry (DSC). In vitro biomineral-binding potential and kinetics were evaluated on hydroxyapatite (HA, a widely used material for orthopedic implant devices) particles. Oxacillin was encapsulated into BBL and used for in vitro evaluation in preventing Staphylococcus aureus biofilm formation.

Results

DSC analysis showed that ALN-TEG-Chol could inhibit the phase transition of liposomes by reducing its cooperativity, yielding liposomes with thermodynamic stability similar to liposomes containing regular cholesterol. BBL showed fast and strong binding ability to HA. Oxacillin-loading BBL demonstrated significantly better preventive efficacy against bacteria colonization when challenged with S. aureus isolate, implying its potential in preventing orthopedic implant associated osteomyelitis.

Conclusions

In this proof of concept study, novel BBL has been successfully developed and validated for reducing the frequency of implantable device-related infections.     

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.     

Engineering of an Inhalable DDA/TDB Liposomal Adjuvant: A Quality-by-Design Approach Towards Optimization of the Spray Drying Process

Purpose

The purpose of this study was to identify and optimize spray drying parameters of importance for the design of an inhalable powder formulation of a cationic liposomal adjuvant composed of dimethyldioctadecylammonium (DDA) bromide and trehalose-6,6′-dibehenate (TDB).

Methods

A quality by design (QbD) approach was applied to identify and link critical process parameters (CPPs) of the spray drying process to critical quality attributes (CQAs) using risk assessment and design of experiments (DoE), followed by identification of an optimal operating space (OOS). A central composite face-centered design was carried out followed by multiple linear regression analysis.

Results

Four CQAs were identified; the mass median aerodynamic diameter (MMAD), the liposome stability (size) during processing, the moisture content and the yield. Five CPPs (drying airflow, feed flow rate, feedstock concentration, atomizing airflow and outlet temperature) were identified and tested in a systematic way. The MMAD and the yield were successfully modeled. For the liposome size stability, the ratio between the size after and before spray drying was modeled successfully. The model for the residual moisture content was poor, although, the moisture content was below 3% in the entire design space. Finally, the OOS was drafted from the constructed models for the spray drying of trehalose stabilized DDA/TDB liposomes.

Conclusions

The QbD approach for the spray drying process should include a careful consideration of the quality target product profile. This approach implementing risk assessment and DoE was successfully applied to optimize the spray drying of an inhalable DDA/TDB liposomal adjuvant designed for pulmonary vaccination.

Quantitative <Emphasis Type="BoldItalic">In Vitro</Emphasis> Assessment of Liposome Stability and Drug Transfer Employing Asymmetrical Flow Field-Flow Fractionation (AF4)

Purpose

In the present study we introduce an efficient approach for a size-based separation of liposomes from plasma proteins employing AF4. We investigated vesicle stability and release behavior of the strongly lipophilic drug temoporfin from liposomes in human plasma for various incubation times at 37°C.

Methods

We used the radioactive tracer cholesteryl oleyl ether (COE) or dipalmitoyl-phosphocholine (DPPC) as lipid markers and ¹⁴C-labeled temoporfin. First, both lipid labels were examined for their suitability as liposome markers. Furthermore, the influence of plasma origin on liposome stability and drug transfer was investigated. The effect of membrane fluidity and PEGylation on vesicle stability and drug release characteristics was also analyzed.

Results

Surprisingly, we observed an enzymatic transfer of ³H-COE to lipoproteins due to the cholesterol ester transfer protein (CETP) in human plasma in dependence on membrane rigidity and were able to inhibit this transfer by plasma preincubation with the CETP inhibitor torcetrapib. This effect was not seen when liposomes were incubated in rat plasma. DPPC labels suffered from hydrolysis effects during preparation and/or storage. Fluid liposomes were less stable in human plasma than their PEGylated analogues or a rigid formulation. In contrast, the transfer of the incorporated drug to lipoproteins was higher for the rigid formulations.

Conclusions

The observed effects render COE-labels questionable for in vivo studies using CEPT-rich species. Here, choline labelled ¹⁴C-DPPC was found to be the most promising alternative. Bilayer composition has a high influence on stability and drug release of a liposomal formulation in human plasma.

     

The Shape/Morphology Balance: A Study of Stealth Liposomes via Fractal Analysis and Drug Encapsulation

Purpose

Fractal analysis was used as a tool in order to study the morphological characteristics of PEGylated liposomes. We report on the morphological characteristics of stealth liposomes composed of DPPC and DPPE-PEG 3000 in two dispersion media using fractal analysis.

Methods

Light scattering techniques were used in order to elucidate the size, the morphology and the surface charge of PEGylated liposomes as a function of PEGylated lipid concentration and temperature. Fluorescence spectroscopy studies revealed a microenvironment of low polarity inside the liposomal membranes.

Results

All formulations were found to retain their physicochemical characteristics for at least 3 weeks. The hydrodynamic radii (R_h) of stealth liposomes were stable in the process of heating up to 50°C; while the fractal dimension values (d_f) which correspond to their morphology, have been changed during heating. Hence, these results are a first indication of the presence of a heterogeneous microdomain structure of the stealth liposomal system. The amphiphilic drug indomethacin (IND) was successfully encapsulated within the liposomes and led to an increased size of stealth liposomes, while the morphology of liposomal vectors changed significantly at the highest molar ratio of PEGylated lipid.

Conclusions

We can state that this approach can promote a new analytical concept based on the morphological characteristics and quantify the shape of drug carriers complementary to that of the conventional analytical techniques.     

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.     

Moisture Uptake of Polyoxyethylene Glycol Glycerides Used as Matrices for Drug Delivery: Kinetic Modelling and Practical Implications

Purpose

Gelucire 50/13, a polyoxyethylene glycol glyceride mixture, has been widely used in drug delivery, but its moisture uptake behaviour is still poorly understood. In this study, the effects of relative humidity, temperature, and drug incorporation on the moisture uptake of Gelucire are reported in relation to their practical implications for preparation of solid dispersions using this material.

Methods

DVS combined with kinetics modelling was used as the main experimental method to study the moisture uptake behaviour of Gelucire. Thermal and microscopic methods were employed to investigate the effect of moisture uptake on the physical properties of the material and drug loaded solid dispersions.

Results

The moisture uptake by Gelucire 50/13 is temperature and relative humidity dependent. At low temperatures and low relative humidities, moisture sorption follows a GAB model. The model fitting indicated that at high relative humidities the sorption is a complex process, potentially involving PEG being dissolved and the PEG solution acting as solvent to dissolve other components.

Conclusion

Careful control of the storage and processing environmental conditions are required when using Gelucire 50/13. The incorporation of model drugs not only influences the moisture uptake capacity of Gelucire 50/13 but also the solidification behaviour.     

A Cell Assay for Detecting Anti-PEG Immune Response against PEG-Modified Therapeutics

Purpose

Immunogenicity of PEGylated proteins and nanomedicines represents a potential impediment against their development and use in clinical settings. The purpose of this study is to develop a method for detecting anti-PEG immunity of PEGylated proteins and/or nanomedicines using flow cytometry.

Methods

The binding of fluorescence-labeled mPEG-modified liposomes to HIK-G11 cells, PEG-specific hybridoma cells, or spleen cells was evaluated by flow cytometry for detecting immunogenicity of PEGylated therapeutics.

Results

The fluorescence-labeled methoxy PEG (mPEG)-modified liposomes were efficiently bound to HIK-G11 cells. Such staining with fluorescence-labeled mPEG-modified liposomes was significantly inhibited in the presence of either non-labeled mPEG-modified liposomes or mPEG-modified ovalbumin (OVA) but not polyglycerol-modified liposomes. In addition, we found that mPEG-modified liposomes, highly immunogenic, caused proliferation of PEG-specific cells, while hydroxyl PEG-modified liposomes, less immunogenic, scarcely caused. Furthermore, after intravenous injection of mPEG-modified liposomes, the percentage of PEG-specific cells in the splenocytes, as determined by flow cytometry, corresponded well with the production level of anti-PEG antibodies, as determined by ELISA.

Conclusions

PEG-specific B cell assay we introduced may become a useful method to detect an anti-PEG immune response against PEGylated therapeutics and clarify the mechanism for anti-PEG immune responses.