Novel liposome-based formulations for prolonged delivery of proteins and vaccines

Abstract

Two strategies for increasing liposome stability in vivo are described in this review. The first strategy involves the encapsulation of liposomes within polymeric microcapsules of alginate-poly(L-lysine) that retained the liposomes inside but allowed the outward diffusion of proteins of 100 kDa or less, once they were released from the encapsulated liposomes. In vivo studies revealed that the microencapsulated liposome systems (MELs) extended the delivery of a model antigen, bovine serum albumin (BSA), for more that 80 days, resulting in the prolonged production of high levels of antigen-specific antibodies. The antibody levels were higher that those obtained with rats injected with BSA in complete Freund's adjuvant, or in liposomes. The unique construction of MELs enabled also the enzymatically-triggered pulsatile delivery of proteins from encapsulated liposomes, which was not possible before with liposomes.     

In situ activation of murine macrophages by liposomes containing lymphokines

Murine alveolar and peritoneal macrophages harvested after injection of lymphokines encapsulated within multilamellar phospholipid vesicles (liposomes) were tumoricidal in vitro. The state and degree of activation depended on the route of liposome administration. Activation of peritoneal macrophages was achieved by intraperitoneal injection of liposomes and alveolar macrophages were activated by injecting liposomes intravenously but not intraperitoneally. The in vivo rendering of macrophages with tumoricidal properties might be useful toward destruction of tumor cells in vivo.     

Biotin and glucose dual-targeting,ligand-modified liposomes promote breast tumor-specific drug delivery

Breast cancer is the second leading cause of cancer-related deaths in women. Ligand-modified liposomes are used for breast tumor-specific drug delivery to improve the efficacy and reduce the side effects of chemotherapy; however, only a few liposomes with high targeting efficiency have been developed because the mono-targeting, ligand-modified liposomes are generally unable to deliver an adequate therapeutic dose. In this study, we designed biotin-glucose branched ligand-modified, dual-targeting liposomes (Bio-Glu-Lip) and evaluated their potential as a targeted chemotherapy delivery system in vitro and in vivo. When compared with the non-targeting liposome (Lip), Bio-Lip, and Glu-Lip, Bio-Glu-Lip had the highest cell uptake in 4T1 cells (3.00-fold, 1.60-fold, and 1.95-fold higher, respectively) and in MCF-7 cells (2.63-fold, 1.63-fold, and 1.85-fold higher, respectively). The subsequent cytotoxicity and in vivo assays further supported the dual-targeting liposome is a promising drug delivery carrier for the treatment of breast cancer.     

A Liposomal Drug Platform Overrides Peptide Ligand Targeting to a Cancer Biomarker,Irrespective of Ligand Affinity or Density

One method for improving cancer treatment is the use of nanoparticle drugs functionalized with targeting ligands that recognize receptors expressed selectively by tumor cells. In theory such targeting ligands should specifically deliver the nanoparticle drug to the tumor, increasing drug concentration in the tumor and delivering the drug to its site of action within the tumor tissue. However, the leaky vasculature of tumors combined with a poor lymphatic system allows the passive accumulation, and subsequent retention, of nanosized materials in tumors. Furthermore, a large nanoparticle size may impede tumor penetration. As such, the role of active targeting in nanoparticle delivery is controversial, and it is difficult to predict how a targeted nanoparticle drug will behave in vivo. Here we report in vivo studies for α_vβ₆-specific H2009.1 peptide targeted liposomal doxorubicin, which increased liposomal delivery and toxicity to lung cancer cells in vitro. We systematically varied ligand affinity, ligand density, ligand stability, liposome dosage, and tumor models to assess the role of active targeting of liposomes to α_vβ₆. In direct contrast to the in vitro results, we demonstrate no difference in in vivo targeting or efficacy for H2009.1 tetrameric peptide liposomal doxorubicin, compared to control peptide and no peptide liposomes. Examining liposome accumulation and distribution within the tumor demonstrates that the liposome, and not the H2009.1 peptide, drives tumor accumulation, and that both targeted H2009.1 and untargeted liposomes remain in perivascular regions, with little tumor penetration. Thus H2009.1 targeted liposomes fail to improve drug efficacy because the liposome drug platform prevents the H2009.1 peptide from both actively targeting the tumor and binding to tumor cells throughout the tumor tissue. Therefore, using a high affinity and high specificity ligand targeting an over-expressed tumor biomarker does not guarantee enhanced efficacy of a liposomal drug. These results highlight the complexity of in vivo targeting.     

Large anti-HER2/neu liposomes for potential targeted intraperitoneal therapy of micrometastatic cancer

Effective targeting and killing of intraperitoneally disseminated micrometastases remains a challenge.

Objective/Methods:?In this work, we evaluated the potential of antibody-labeled PEGylated large liposomes as vehicles for direct intraperitoneal (i.p.) drug delivery with the aim to enhance the tumor-to-normal organ ratio and to improve the bioexposure of cancer cells to the delivered therapeutics while shifting the toxicities toward the spleen. These targeted liposomes are designed to combine: (1) specific targeting to and internalization by cancer cells mediated by liposome-conjugated tumor-specific antibodies, (2) slow clearance from the peritoneal cavity, and (3) shift of normal organ toxicities from the liver to the spleen due to their relatively large size.

Results:?Conjugation of anti-HER2/neu antibodies to the surface of large (approximately 600?nm in diameter) PEGylated liposomes results in fast, specific binding of targeted liposomes to cancer cells in vitro, followed by considerable cellular internalization. In vivo, after i.p. administration, these liposomes exhibit fast, specific binding to i.p. cancerous tumors. Large liposomes are slowly cleared from the peritoneal cavity, and they exhibit increased uptake by the spleen relative to the liver, while targeted large liposomes demonstrate specific tumor uptake at early times. Although tissue and tumor uptake are greater for cationic liposomes, the tumor-to-liver and spleen-to-liver ratios are similar for both membrane compositions, suggesting a primary role for the liposome’s size, compared to the liposome’s surface charge.

Conclusions:?The findings of this study suggest that large targeted liposomes administered i.p. could be a potent drug-delivery strategy for locoregional therapy of i.p. micrometastatic tumors.     

LIPOSOMES CONTAINING DOPAMINE ENTRAPPED IN RESPONSE TO TRANSMEMBRANE AMMONIUM SULFATE GRADIENT AS CARRIER SYSTEM FOR DOPAMINE DELIVERY INTO THE BRAIN OF PARKINSONIAN MICE

The active loading of liposomes with dopamine in response to an ammonium sulfate gradient was studied. This method can be regarded as a mean to more efficiently improve the liposomal dopamine/lipids ratio in comparison to conventional methods of liposome preparation. Trapping efficiency of dopamine into liposomes exhibiting a transmembrane ammonium sulfate gradient was shown to be dependent on liposome lipid composition, lipid concentration and temperature. Dopamine-containing liposomes with α-tocopherol in the lipid bilayer were shown to be stable at least for three weeks. It has been found that intraperitoneal (i.p.) administration of conventionally prepared dopamine-containing liposomes as well as liposomes with increased dopamine/lipid ratio may efficiently suppress the expression of parkinsonian symptoms in C57BL/6 mice with experimental parkinsonian syndrome. On the other hand, only through increasing of liposomal dopamine/lipid ratio the complete compensation of dopamine deficiency in the mice brain was achieved. The obtained data may be considered as biochemical evidence in favor of liposomes' ability to act as a carrier system for the delivery of dopamine into the brain.     

Structure and stability of anionic liposomes complexes with PEG-chitosan branched copolymer

Application of branched copolymers of polyethylene glycol and chitosan (PEG-chitosan) as stabilizing agents for anionic liposomes was shown to improve considerably liposomes storage stability. In the course of the work, an efficient and convenient approach to synthesis of PEG-chitosan copolymers through chemical modification of chitosan amino groups with monomethoxy-PEG-N-hydroxysuccinimidyl succinate (mPEG-suc-NHS) was developed. Chitosan with varying degree of PEGylation were obtained and used as stabilizing agents for anionic liposomes prepared of dipalmitoylphosphatidylcholine-cardiolipin, 80/20 by weight. The molecular mechanism of complex formation between the anionic liposomes and PEG-chitosan was studied by methods of FTIR spectroscopy and dynamic light scattering. Phosphate and carbonyl groups were found to be the main sites of the aminopolysaccharide binding. Stabilization of the complexes is mainly achieved through electrostatic interactions between anionic groups of cardiolipin and free amino groups of PEG-chitosan. The method of liposome stabilization is promising for the development of new drug delivery systems.     

Dual-targeting for brain-specific liposomes drug delivery system: Synthesis and preliminary evaluation

The treatment of glioma has become a great challenge because of the existence of brain barrier (BB). In order to develop an efficient brain targeting drug delivery system to greatly improve the brain permeability of anti-cancer drugs, a novel brain-targeted glucose-vitamin C (Glu-Vc) derivative was designed and synthesized as liposome ligand for preparing liposome to effectively deliver paclitaxel (PTX). The liposome was prepared and its particle size, zeta potential, encapsulation efficiency, release profile, stability, hemolysis and cytotoxicity were also characterized. What’s more, the cellular uptake of CFPE-labeled Glu-Vc-Lip on GLUT₁- and SVCT₂-overexpressed C6 cells was 4.79-, 1.95-, 4.00- and 1.53-fold higher than that of Lip, Glu-Lip, Vc-Lip and Glu?+?Vc-Lip. Also, the Glu-Vc modified liposomes showed superior targeting ability in vivo evaluation compared with naked paclitaxel, non-coated, singly-modified and co-modified by physical blending liposomes. The relative uptake efficiency was enhanced by 7.53 fold to that of naked paclitaxel, while the concentration efficiency was up to 7.89 times. What’s more, the Glu-Vc modified liposomes also displayed the maximum accumulation of DiD-loaded liposomes at tumor sites with the strongest fluorescence in the brain in vivo imaging. Our results suggest that chemical modification of liposomes with warheads of glucose and vitamin C represents a promising and efficient strategy for the development of brain-specific liposomes drug delivery system by utilizing the endogenous transportation mechanism of the warheads.     

Effect of pH on the Control Release of Microencapsulated Dye in Lecithin Liposomes. II

Abstract

The objective of our work has been the microencapsulation of dyes with lecithin from soybean, with the formation of liposomes, as a substitute for synthetic auxiliaries so as to improve the quality of the effluent. Current scenarios promote the disintegration and leakage of the liposomes, such as, changes in temperature, pH, and the use of surfactants. Since dyeing process is a mix of all these parameters, we pretended to study each one separately. Changes in pH at constant temperature induce a release of dye similar with changes in temperature. In acid conditions, we found a very fast initial dye release which doesn't occur in basic conditions. Using carboxyfluorescein, as a pH fluorescence probe, we concluded that the liposome membrane doesn't protect the liposome interior from changes on the external pH.     

Pulmonary Delivery of Liposomal Drugs

Abstract

This overview will discuss our studies of liposomes aerosols to treat diseases of the lung and will entail (i) formulation and characterization of liposome aerosols, including dry liposome powder aerosols, (ii) modulation of the pharmacokinetic profile of liposomal drugs delivered by aerosol or intratracheal instillation, (iii) liposome-alveolar macrophage interactions in vitro and in vivo, and (iv) safety of liposome aerosols in vivo in mice, sheep and healthy human volunteers. Water-soluble agents can be retained in liposomes during aerosolization with air-pressure nebulizers within certain limitations of liposome composition, size, and operating conditions. Dry powder liposome aerosols have been formulated and deliver water-soluble encapsulated substances efficiently. Pharmacokinetic profiles of liposomal drugs delivered via intratracheal instillation exhibit typical slow release plasma profiles indicating that the carrier is the rate-limiting barrier for release. Accordingly, pulmonary mean residence times are significantly prolonged and systemic concentrations remain low. Liposomes do not inhibit the phagocytic activity of alveolar macrophages in vitro and in vivo, have no apparent histopathologic effects on lung architecture even after chronic administration, and do not alter dynamic compliance, lung resistance, p_aO₂ and p_aCO₂ in awake, unanesthetized sheep and in healthy human volunteers. In conclusion, liposomes are a promising innocuous aerosol delivery system for drugs to achieve prolonged localized drug concentrations in the lung or intracellular drug targeting to alveolar macrophages.     

Therapeutic dendritic cell vaccine preparation using tumor RNA transfection: A promising approach for the treatment of prostate cancer

Background

Electroporation is an established technique for enhancing plasmid delivery to many tissues in vivo, including the skin. We have previously demonstrated efficient delivery of plasmid DNA to the skin utilizing a custom-built four-plate electrode. The experiments described here further evaluate cutaneous plasmid delivery using in vivo electroporation. Plasmid expression levels are compared to those after liposome mediated delivery.

Methods

Enhanced electrically-mediated delivery, and less extensively, liposome complexed delivery, of a plasmid encoding the reporter luciferase was tested in rodent skin. Expression kinetics and tissue damage were explored as well as testing in a second rodent model.

Results

Experiments confirm that electroporation alone is more effective in enhancing reporter gene expression than plasmid injection alone, plasmid conjugation with liposomes followed by injection, or than the combination of liposomes and electroporation. However, with two time courses of multiple electrically-mediated plasmid deliveries, neither the levels nor duration of transgene expression are significantly increased. Tissue damage may increase following a second treatment, no further damage is observed after a third treatment. When electroporation conditions utilized in a mouse model are tested in thicker rat skin, only higher field strengths or longer pulses were as effective in plasmid delivery.

Conclusion

Electroporation enhances reporter plasmid delivery to the skin to a greater extent than the liposome conjugation method tested. Multiple deliveries do not necessarily result in higher or longer term expression. In addition, some impact on tissue integrity with respect to surface damage is observed. Pulsing conditions should be optimized for the model and for the expression profile desired.     

Stability of association of 1-O-octadecyl-2-O-methyl-sn-glycero-3-phosphocholine with liposomes is composition dependent

The ether lipid, 1-O-octadecyl-2-O-methyl-sn-glycero-3-phosphocholine (ET-18-OCH₃), has anticancer activity, but it has serious side-effects, including hemolysis, which prevent its optimal use. We surmised if ET-18-OCH₃ could be stably associated with liposomes, less free ET-18-OCH₃ would be available for lytic interaction with red cells. Liposome composition variables investigated included acyl chain saturation, phospholipid head group and mole ratio of Chol and ET-18-OCH₃. It was found that attenuation of hemolysis was strongly liposome composition dependent. Some ET-18-OCH₃ liposome compositions were minimally hemolytic. For example, whereas the HI₅ (drug concentration required to cause 5% human red cell lysis) was 5–6 μM for free ET-18-OCH₃, it was approximately 250 μM for DOPC (dioleoylphosphatidylcholine):Chol (cholesterol):DOPE-GA (glutaric acid derivatized DOPE):ET-18-OCH₃, (4:3:1:2) and 640 μM for DOPE (dioleyolphosphatidylethanolamine):Chol:DOPE-GA:ET-18-OCH₃ (4:3:1:2) liposomes. Efflux of carboxyfluorescein (CF) from liposomes and Langmuir trough determinations of mean molecular area of lipids in monolayers (MMAM) were used as indicators of membrane packing and stability. Incorporation of ET-18-OCH₃ in liposomes reduced the MMAM. Reduction in CF permeation was correlated with reduction in hemolysis. The most stable liposomes included components, such as cholesterol, DOPC and DOPE, which have complementary shapes to ET-18-OCH₃.     

Combinatorial Synthesis of and High-throughput Protein Release from Polymer Film and Nanoparticle Libraries

Polyanhydrides are a class of biomaterials with excellent biocompatibility and drug delivery capabilities. While they have been studied extensively with conventional one-sample-at-a-time synthesis techniques, a more recent high-throughput approach has been developed enabling the synthesis and testing of large libraries of polyanhydrides¹. This will facilitate more efficient optimization and design process of these biomaterials for drug and vaccine delivery applications. The method in this work describes the combinatorial synthesis of biodegradable polyanhydride film and nanoparticle libraries and the high-throughput detection of protein release from these libraries. In this robotically operated method (Figure 1), linear actuators and syringe pumps are controlled by LabVIEW, which enables a hands-free automated protocol, eliminating user error. Furthermore, this method enables the rapid fabrication of micro-scale polymer libraries, reducing the batch size while resulting in the creation of multivariant polymer systems. This combinatorial approach to polymer synthesis facilitates the synthesis of up to 15 different polymers in an equivalent amount of time it would take to synthesize one polymer conventionally. In addition, the combinatorial polymer library can be fabricated into blank or protein-loaded geometries including films or nanoparticles upon dissolution of the polymer library in a solvent and precipitation into a non-solvent (for nanoparticles) or by vacuum drying (for films). Upon loading a fluorochrome-conjugated protein into the polymer libraries, protein release kinetics can be assessed at high-throughput using a fluorescence-based detection method (Figures 2 and 3) as described previously¹. This combinatorial platform has been validated with conventional methods² and the polyanhydride film and nanoparticle libraries have been characterized with ¹H NMR and FTIR. The libraries have been screened for protein release kinetics, stability and antigenicity; in vitro cellular toxicity, cytokine production, surface marker expression, adhesion, proliferation and differentiation; and in vivo biodistribution and mucoadhesion^1-11. The combinatorial method developed herein enables high-throughput polymer synthesis and fabrication of protein-loaded nanoparticle and film libraries, which can, in turn, be screened in vitro and in vivo for optimization of biomaterial performance.     

<Emphasis Type="Italic">In vitro</Emphasis> and <Emphasis Type="Italic">In vivo</Emphasis> Studies on a Novel Bioadhesive Colloidal System: Cationic Liposomes of Ibuprofen

The objective of this study was to develop an ocular drug delivery system built on the cationic liposomes, a novel bioadhesive colloidal system, which could enhance the precorneal residence time, ocular permeation, and bioavailability of ibuprofen. The optimal formulation of cationic liposomes prepared by ethanol injection method was ultimately confirmed by an orthogonal L₉ (3³) test design. In addition, γ-scintigraphic technology and the microdialysis technique were utilized in the assessment of in vivo precorneal retention capability and ocular bioavailability individually. In the end, we acquired the optimal formulation of ibuprofen cationic liposomes (Ibu-CL) by orthogonal test design, and the particle size and entrapment efficiency (EE%) were 121.0 ± 3.5 nm and 72.9 ± 3.4%, respectively. In comparison to ibuprofen eye drops (Ibu-ED), Ibu-CL could significantly prolong the T _max to 100 min and the AUC to 1.53-folds, which indicated that the Ibu-CL could improve the precorneal retention time and bioavailability of ibuprofen. Consequently, these outcomes designated that the ibuprofen cationic liposomes we researched probably are a promising application in ocular drug delivery system.     

A simple method for producing a technetium-99m-labeled liposome which is stable In Vivo

A new method for labeling preformed liposomes with technetium-99m (^99mTc) has been developed which is simple to perform and stable in vivo. Previous ^99mTc-liposome labels have had variable labeling efficiencies and stability. This method consistently achieves high labeling efficiencies (> 90%) with excellent stability. A commercially available radiopharmaceutical kit—hexamethylpropyleneamine oxime (HM-PAO)—is reconstituted with ^99mTcO^−₄ and then incubated with preformed liposomes that encapsulate glutathione. The incubation takes only 30 min at room temperature. Liposomes that co-encapsulate other proteins such as hemoglobin or albumin, in addition to glutathione, also label with high efficiency. Both in vitro and in vivo studies indicate good stability of this label. Rabbit images show significant spleen and liver uptake at 2 and 20 h after liposome infusion without visualization of thyroid, stomach or bladder activity.This labeling method can be used to study the biodistribution of a wide variety of liposome preparations that are being tested as novel drug delivery systems. This method of labeling liposomes with ^99mTc may also have applications in diagnostic imaging.     

Liposomes to Target Peripheral Neurons and Schwann Cells

While a wealth of literature for tissue-specific liposomes is emerging, optimal formulations to target the cells of the peripheral nervous system (PNS) are lacking. In this study, we asked whether a novel formulation of phospholipid-based liposomes could be optimized for preferential uptake by microvascular endothelia, peripheral neurons and Schwann cells. Here, we report a unique formulation consisting of a phospholipid, a polymer surfactant and cholesterol that result in enhanced uptake by targeted cells. Using fluorescently labeled liposomes, we followed particle internalization and trafficking through a distinct route from dextran and escape from degradative compartments, such as lysosomes. In cultures of non-myelinating Schwann cells, liposomes associate with the lipid raft marker Cholera toxin, and their internalization is inhibited by disruption of lipid rafts or actin polymerization. In contrast, pharmacological inhibition of clathrin-mediated endocytosis does not significantly impact liposome entry. To evaluate the efficacy of liposome targeting in tissues, we utilized myelinating explant cultures of dorsal root ganglia and isolated diaphragm preparations, both of which contain peripheral neurons and myelinating Schwann cells. In these models, we detected preferential liposome uptake into neurons and glial cells in comparison to surrounding muscle tissue. Furthermore, in vivo liposome administration by intramuscular or intravenous injection confirmed that the particles were delivered to myelinated peripheral nerves. Within the CNS, we detected the liposomes in choroid epithelium, but not in myelinated white matter regions or in brain parenchyma. The described nanoparticles represent a novel neurophilic delivery vehicle for targeting small therapeutic compounds, biological molecules, or imaging reagents into peripheral neurons and Schwann cells, and provide a major advancement toward developing effective therapies for peripheral neuropathies.     

Polymer-Grafted Liposomes: Physical Basis for the “Stealth” Property

Abstract

Polymer-bearing lipids have recently been incorporated into liposomes that are used in in vivo drug delivery. This strategy has improved the liposome's ability to avoid the reticuloendothelial system and has thereby increased its circulation time in the bloodstream. In order to understand the physical basis for this, so called, Stealth® effect, we have begun a series of studies that characterize the surface structure, interactive properties and in vivo performance of the polymer-bearing, Stealth lipids. For a 1900 g/mol polyethylene glycol (PEG) moiety, we have used x-ray diffraction and micropipet manipulation methods to show that, (i) the polymer chains extend ～50Å out from the lipid bilayer surface; (ii) this surface polymer exerts a significant long range mutual repulsion between adjacent bilayers that prevents bilayer-bilayer adhesion. Furthermore, the measured polymer extension and repulsive pressure are well modelled by polymer scaling laws. These results imply that the interaction of macromolecules and cellular surfaces with the Stealth liposome is probably limited to a distance of ～50Å from the liposome surface. We conclude that the origin of the Stealth effect lies in a steric stabilization mechanism. By using fluorescence video microscopy to observe implanted tumor tissue, we have also shown that fluorescent Stealth liposomes extravasate through the leaky vessel walls of tumors. This method allows us to characterize, in real time, the accumulation of liposomes and release of drug at an implanted tumor site.     

Targeting of Liposomes Within Cardiovascular System

Abstract

Our studies on the targeting of liposomes and liposome-associated pharmaceuticals within the cardiovascular system are reviewed. The delivery of diagnostic and therapeutic agents in plain liposomes, immunoliposomes, long-circulating liposomes and long-circulating immunoliposomes into the sites of vascular injuries and myocardial infarction is discussed. In vitro, ex vivo, and in vivo experiments present a general view on the advantages and limitations of using liposome-mediated targeting. Liposomes capable of targeting pathological areas of the blood vessel wall both, in vitro and ex vivo are described, as well as liposome able to be internalized by normal endothelial cells. Liposome-mediated drug targeting to compromised myocardium is reviewed with a primary impact on liposomes with anti-cardiac myosin antibodies. Targeted visualization of myocardial infarction with diagnostic liposomes is discussed. Efficient accumulation of long-circulating immunoliposomes in the infarct zone is demonstrated, and a relative importance of different variables, such as liposome size, targetability, and prolonged circulation time, for target accumulation is analyzed. The use of immunoliposomes for targeted sealing of hypoxia-caused damages in plasmic membranes of cardiocytes is considered as a new approach in the therapeutic use of liposomes.     

Femtosecond laser-based mutagenesis strategy for micronomicin production enhancement of Micromonospora sagamiensis ATCC 21826

In this work, a mutant MX3004 with improved micronomicin (MCR) production was derived from Micromonospora sagamiensis ATCC21826, which was treated with femtosecond laser under the optimized irradiation conditions of 75 mW and 180 s, with a maximum of positive mutation rate of 17.8 % and the mortality rate of 69.2 %. A novel high-throughput method was established using microplate reader by quantifying the concentration of MCR for efficient screening of positive mutant from large numbers of mutants. Consequently, MX3004 displayed the highest MCR production capacity of 126 U/ml and a stable heredity (ten generations). Moreover, under the optimal fermentation conditions in a 7.5 l fermenter, the MCR production of MX3004 reached the maximum of 263 U/ml, which was increased by 484 % compared with the parent strain. The results suggest that femtosecond laser is a suitable method for the MCR production improvement and the screening method has a great potential application for aminoglycoside antibiotic production.     

Use of β-galactosidase liposome model as a novel method to screen freeze-drying cryoprotectants

This study developed a novel method of screening cryoprotectants used to improve the survivability of lyophilized Lactobacillus helveticus. To develop a liposome encapsulated β-galactosidase (β-gal) as a cell membrane model, the β-gal liposome was characterized in terms of mean size, poly dispersity index, zeta potential, along with transmission electron microscopy. 800 W of ultrasonic power and 10 min of sonication time were the optimal experimental conditions to obtain the desirable β-gal liposome. Subsequently, different cryoprotectants were mixed with the β-gal liposome during freeze-drying. After freeze-drying, liposomes were hydrolized, and the protective effect of cryoprotectants was assessed as the release rate of encapsulated β-gal. The lowest release rate of β-gal was obtained using 10 mg/100 ml trehalose and 0.2 mg/100 ml hyaluronic acid.