Overcoming neutrophil-induced immunosuppression in postoperative cancer therapy: Combined sialic acid-modified liposomes with scaffold-based vaccines

Immunotherapy is a promising approach for preventing postoperative tumor recurrence and metastasis. However, inflammatory neutrophils, recruited to the postoperative tumor site, have been shown to exacerbate tumor regeneration and limit the efficacy of cancer vaccines. Consequently, addressing postoperative immunosuppression caused by neutrophils is crucial for improving treatment outcomes. This study presents a combined chemoimmunotherapeutic strategy that employs a biocompatible macroporous scaffold-based cancer vaccine (S-CV) and a sialic acid (SA)-modified, doxorubicin (DOX)-loaded liposomal platform (DOX@SAL). The S-CV contains whole tumor lysates as antigens and imiquimod (R837, Toll-like receptor 7 activator)-loaded PLGA nanoparticles as immune adjuvants for cancer, which enhance dendritic cell activation and cytotoxic T cell proliferation upon localized implantation. When administered intravenously, DOX@SAL specifically targets and delivers drugs to activated neutrophils in vivo, mitigating neutrophil infiltration and suppressing postoperative inflammatory responses. In vivo and vitro experiments have demonstrated that S-CV plus DOX@SAL, a combined chemo-immunotherapeutic strategy, has a remarkable potential to inhibit postoperative local tumor recurrence and distant tumor progression, with minimal systemic toxicity, providing a new concept for postoperative treatment of tumors.

Liposomes as versatile agents for the management of traumatic and nontraumatic central nervous system disorders:drug stability,targeting efficiency,and safety

Various nanoparticle-based drug delivery systems for the treatment of neurological disorders have been widely studied.However,their inability to cross the blood–brain barrier hampers the clinical translation of these therapeutic strategies.Liposomes are nanoparticles composed of lipid bilayers,which can effectively encapsulate drugs and improve drug delivery across the blood–brain barrier and into brain tissue through their targeting and permeability.Therefore,they can potentially treat traumatic and nontraumatic central nervous system diseases.In this review,we outlined the common properties and preparation methods of liposomes,including thin-film hydration,reverse-phase evaporation,solvent injection techniques,detergent removal methods,and microfluidics techniques.Afterwards,we comprehensively discussed the current applications of liposomes in central nervous system diseases,such as Alzheimer's disease,Parkinson's disease,Huntington's disease,amyotrophic lateral sclerosis,traumatic brain injury,spinal cord injury,and brain tumors.Most studies related to liposomes are still in the laboratory stage and have not yet entered clinical trials.Additionally,their application as drug delivery systems in clinical practice faces challenges such as drug stability,targeting efficiency,and safety.Therefore,we proposed development strategies related to liposomes to further promote their development in neurological disease research.

Lyophilization Process of Hydroxypropyl Tetrahydropyrantriol Liposomes

[Objectives]To enhance the skin permeability of hydroxypropyl tetrahydropyrantriol and provide a reference for the subsequent prevention or treatment of skin aging.[Methods]The lyophilization process of hydroxypropyl tetrahydropyrantriol liposomes was investigated using a single factor method,and a quality evaluation system was established based on the appearance,particle size,PDI,and re-dispersibility of the lyophilized samples.[Results]The lyophilization process of hydroxypropyl tetrahydropyrantriol liposomes was determined by single factor experiments.The pre-freezing period was 16 h at-80℃,the total drying time was 36 h,and the addition of 10%mannitol-sucrose was used as the lyoprotectant.[Conclusions]The product prepared by the lyophilization method exhibits a fluffy and full appearance,with minimal shrinkage and collapse.The volume remains consistent before and after lyophilization,and the re-dispersibility is satisfactory.The re-dissolution process is rapid,and the particle size and polydispersity index(PDI)remain largely unchanged before and after lyophilization.

Leveraging immunoliposomes as nanocarriers against SARS-CoV-2 and its emerging variants

The global COVID-19 pandemic arising from SARS-CoV-2 has impacted many lives,gaining interest worldwide ever since it was first identified in December 2019.Till 2023,752 million cumulative cases and 6.8 million deaths were documented globally.COVID-19 has been rapidly evolving,affecting virus transmissibility and properties and contributing to increased disease severity.The Omicron is themost circulating variant of concern.Although success in its treatment has indicated progress in tackling the virus,limitations in delivering the current antiviral agents in battling emerging variants remain remarkable.With the latest advancements in nanotechnology for controlling infectious diseases,liposomes have the potential to counteract SARS-CoV-2 because of their ability to employ different targeting strategies,incorporating monoclonal antibodies for the active and passive targeting of infected patients.This review will present a concise summary of the possible strategies for utilizing immunoliposomes to improve current treatment against the occurrence of SARSCoV-2 and its variants.

Selective ischemic-hemisphere targeting Ginkgolide B liposomes with improved solubility and therapeutic efficacy for cerebral ischemia-reperfusion injury

Cerebral ischemia-reperfusion injury(CI/RI)remains the main cause of disability and death in stroke patients due to lack of effective therapeutic strategies.One of the main issues related to CI/RI treatment is the presence of the blood-brain barrier(BBB),which affects the intracerebral delivery of drugs.Ginkgolide B(GB),a major bioactive component in commercially available products of Ginkgo biloba,has been shown significance in CI/RI treatment by regulating inflammatory pathways,oxidative damage,and metabolic disturbance,and seems to be a candidate for stroke recovery.However,limited by its poor hydrophilicity and lipophilicity,the development of GB preparations with good solubility,stability,and the ability to cross the BBB remains a challenge.Herein,we propose a combinatorial strategy by conjugating GB with highly lipophilic docosahexaenoic acid(DHA)to obtain a covalent complex GB-DHA,which can not only enhance the pharmacological effect of GB,but can also be encapsulated in liposomes stably.The amount of finally constructed Lipo@GB-DHA targeting to ischemic hemisphere was validated 2.2 times that of free solution in middle cerebral artery occlusion(MCAO)rats.Compared to the marketed ginkgolide injection,Lipo@GB-DHA significantly reduced infarct volume with better neurobehavioral recovery in MCAO rats after being intravenously administered both at 2 h and 6 h post-reperfusion.Low levels of reactive oxygen species(ROS)and high neuron survival in vitro was maintained via Lipo@GB-DHA treatment,while microglia in the ischemic brain were polarized from the pro-inflammatory M1 phenotype to the tissue-repairing M2 phenotype,which modulate neuroinflammatory and angiogenesis.In addition,Lipo@GB-DHA inhibited neuronal apoptosis via regulating the apoptotic pathway and maintained homeostasis by activating the autophagy pathway.Thus,transforming GB into a lipophilic complex and loading it into liposomes provides a promising nanomedicine strategy with excellent CI/RI therapeutic efficacy and industrialization prospects.

Mechanistic engineering of celastrol liposomes induces ferroptosis and apoptosis by directly targeting VDAC2 in hepatocellular carcinoma

Hepatocellular carcinoma(HCC)is one of most common and deadliest malignancies.Celastrol(Cel),a natural product derived from the Tripterygium wilfordii plant,has been extensively researched for its potential effectiveness in fighting cancer.However,its clinical application has been hindered by the unclear mechanism of action.Here,we used chemical proteomics to identify the direct targets of Cel and enhanced its targetability and antitumor capacity by developing a Cel-based liposomes in HCC.We demonstrated that Cel selectively targets the voltage-dependent anion channel 2(VDAC2).Cel directly binds to the cysteine residues of VDAC2,and induces cytochrome C release via dysregulating VDAC2-mediated mitochondrial permeability transition pore(mPTP)function.We further found that Cel induces ROS-mediated ferroptosis and apoptosis in HCC cells.Moreover,coencapsulation of Cel into alkyl glucoside-modified liposomes(AGCL)improved its antitumor efficacy and minimized its side effects.AGCL has been shown to effectively suppress the proliferation of tumor cells.In a xenograft nude mice experiment,AGCL significantly inhibited tumor growth and promoted apoptosis.Our findings reveal that Cel directly targets VDAC2 to induce mitochondria-dependent cell death,while the Cel liposomes enhance its targetability and reduces side effects.Overall,Cel shows promise as a therapeutic agent for HCC.

Preparation Process of Hydroxypropyl Tetrahydropyrantriol Liposomes

[Objectives] To explore the optimal process for preparing hydroxypropyl tetrahydropyrantriol liposomes. [Methods] A refractive index method was used to determine the content of hydroxypropyl tetrahydropyrantriol. Using particle size distribution and encapsulation rate as evaluation indicators, the effects of hydration time, ratio of organic phase to aqueous phase, granulation method, as well as thin film dispersion and reverse evaporation methods on liposomes preparation were investigated, and the optimal preparation method was selected. Single factor experiments were used to screen the drug phospholipid ratio, ultrasound time, and phospholipid cholesterol ratio, and the preparation process was optimized through orthogonal experiments. [Results] The optimal process of preparing hydroxypropyl tetrahydropyrantriol liposomes was as below: 1 : 10 of drug phospholipid ratio, 6 min of ultrasound time, 4 : 1 of phospholipid cholesterol ratio, (60.94%±7.24%) of entrapment efficiency, (86.44±6.08) nm of particle size, (0.195±0.077) of PDI. [Conclusions] The optimal preparation process of hydroxypropyl tetrahydropyrantriol liposomes selected by orthogonal experiment could effectively improve the encapsulation efficiency of hydroxypropyl tetrahydropyranotriol and reduce particle size. Moreover, the method was stable and reliable.

Doxorubicin Stealth Liposomes Prepared with PEG-Distearoyl Phosphatidylethanolamine and Distribution as well as Antitumor Activity in Mice

目的：研制出能够逃避体内网状内皮细胞的阿霉素隐形脂质体，并考察其在生物体内的分布以及比较阿霉素隐形脂质体与阿霉素普通脂质体的抗肿瘤活性。方法：将聚乙二醇－二硬脂酰磷脂酰乙醇胺（PEG－DSPE）同磷脂酰胆碱和胆固醇材料加在一起，用硫酸铵梯度法制备阿霉素隐形脂质体，同法制备阿霉素普通脂质体（但脂膜中不含PEG－DSPE）；通过尾静脉注射给药，比较隐形脂质体阿霉素、普通脂质体阿霉素和游离型阿霉素（盐酸阿霉素注射液）给药后在小鼠各主要脏器组织和血液中的分布情况；采用动物移植性肿瘤实验法，用H22小鼠肝癌细胞接种于小鼠右侧腋皮下形成实体瘤，考察阿霉素隐形脂质体和普通脂质体给药后对实体瘤的瘤重抑制率。结果：通过硫酸铵梯度法制备出了包封率高达95％的阿霉素隐形脂质体；同游离型阿霉素和普通脂质体阿霉素相比，隐形脂质体阿霉素在血液中浓度显著提高，循环时间显著延长，在心脏中分布的浓度显著降低；按5mg·kg^-1剂量治疗，第二天给药和第七天给药治疗方案，阿霉素隐形脂质体给药组的瘤重抑制率均显著高于普通脂质体给药组的瘤重抑制率；按10mg·kg^-1剂量治疗，隐形脂质体给药组的瘤重抑制率比普通脂质体给药组的瘤重抑制率稍高。结论：用普通脂质体给药相比，隐形脂质体阿霉素给药后延长了其在小鼠血液中的循环时间，说明经过PEG－DSPE修饰后的脂质体有逃避网状内皮细胞吞噬的功能（隐形），并且隐形脂质体阿霉素的抗肿瘤活性显著地提高。

Preparation, Stability and Immunoenhancement of APS (Astragalus polysaccharide) Liposomes

Six factors and 10 levels of each factor were selected by using the (uniform design method( with the aid of the computer for preparing APS liposomes. The optimal procedure for preparing APS liposomes was established and it can suit the large scale production in a pharmaceutical factory. The shelf-life of APS liposomes at 20℃ is 1.46 years. Diameters of the vesicles ( > 90% ) in APS liposomes are less than 1 μm, and the system is stable. At 40℃ the diameters of vesicles were not changed in three months. Pharmacological experiments revealed that APS liposomes exerted a strong immunoenhancement in mice. Studies in this paper established a foundation for the production and the clinical application of APS liposomes.

Effect of the structure of ginsenosides on the in vivo fate of their liposomes

To utilize themultiple functions and give full play of ginsenosides,a variety of ginsenosides with different structures were prepared into liposomes and evaluated for their effect on the stability,pharmacokinetics and tumor targeting capability of liposomes.The results showed that the position and number of glycosyl groups of ginsenosides have significant effect on the in vitro and in vivo properties of their liposomes.The pharmacokinetics of ginsenosides liposomes indicated that the C-3 sugar group of ginsenosides is beneficial to their liposomes for longer circulation in vivo.The C-3 and C-6 glycosyls can enhance the uptake of their liposomes by 4T1 cells,and the glycosyls at C-3 position can enhance the tumor active targeting ability significantly,based on the specific binding capacity to Glut 1 expressed on the surface of 4T1 cells.According to the results in the study,ginsenoside Rg3 and ginsenoside Rh2 are potential for exploiting novel liposomes because of their cholesterol substitution,long blood circulation and tumor targeting capabilities.The results provide a theoretical basis for further development of ginsenoside based liposome delivery systems.

In vitro characterization of two new doxorubicin liposomes modified with argine-glycine-aspartic acid tripeptide or glycine-argine-glycine-aspartic acid-serine pentapeptide

Aim Peptides as ligands have shown the active targeting properties to the receptors like integrins, a family of receptors over-expressed in cancers. The present study was to develop and characterize two peptides modified drug-containing liposomes. Methods Argine-glycine-aspartic acid （RGD） tripeptide and glycine-argine-glycine-aspartic acid-serine （GRGDS） pentapeptide were used for modifications on the doxorubicin-loaded sterically stabilized liposomes （SSL-doxorubicin） for the liposome preparation, RGD-SSL-doxorubicin and GRGDS-SSL-doxorubicin, respectively. Characterizations were performed by measurements of the encapsulation efficiency, particle size and zeta potential, release rates in a simulated in vivo environment, and cytotoxicity to ovarian cancer cells. Cell uptake was investigated by flow cytometry and confocal microscopy methods. Results All encapsulation efficiencies of the liposomes were above 95%, and the modifications using RGD or GRGDS did not affect the final encapsulation efficiency. Average particle sizes of the liposomes Were in the range between 105.7 ± 3.5 nm and 130.5 ± 3.0 nm, and zeta potential values were between -3.3 ± 0.3 and -6.1 ± 0.3 mV. Approximately 2/5 of doxorubicin was released from liposomes before 12 h in the simulated in vivo environment containing fetal bovine serum. Inhibitory rates to cancer cells of the modified liposomes were slightly lower as compared to free doxorubicin. Similar phenomena were observed in the uptake measured by flow cytometry and confocal assay. After uptake applying various formulations on the cancer cells, doxorubicin was mainly distributed in the nuclei of SKOV-3 cells. Conclusion Two new doxorubicin-contained liposomes were successfully prepared and modified with argine-glycine-aspartic acid （RGD） tripeptide and glycine-argine-glycine- aspartic acid-serine （GRGDS） pentapeptide. In vitro characterization indicated that modifications did not alter significantly the properties of the sterically stabilized liposomes.

Pharmaceutical Characteristics of Daunorubicin Stealth Liposomes

This report studied on pharmaceutical characteristics of the stealth liposome containing dau-norubicin (DNR). The shape, size, entrapment efficiency and stability of the daunorubicin stealth liposomes (DNRSL) were examined. Visible spectrophotometry and the HPLC method were established for determination of the DNR in the DNRSL. The release of DNR from DNRSL in HBS (pH 7.5) and rat serum at 37 oC were examined. The results showed that the DNRSL had high entrapment efficiency (>85%), small size and slow release.

Preparation of Liposomes with Palmitic-Antibody andTargeting in Vitro

An anti-trichomonas vaginalis monoclonal antiboody was derivatized with palmitic acid using an activated ester of N-hydroxysuccinimide About 50% of the re-sulting antibody could be incorporated into liposomes.The liposomes showed specific binding to T. vaginalis by IFA and cytotoxicity tests. These results clearly demonstrated the effectiveness of targeting of liposomes modified by monoclonal antibody in vitro.

In vitro investigation of RGD-modified stabilized cationic liposomes as non-viral vehicle for siRNA delivery

In this study, the novel RGD-modified stabilized cationic liposomes were developed as the delivery vehicle for siRNA targeting human MDR1 gene. The complex of cationic liposomes and siRNA, RGD-Lipo-siRNA, was prepared with a narrow size distribution below 200 nm. It was shown that the encapsulated siRNA in the liposomes could be effectively protected from serum degradation. Also, enhanced cell binding and intracellular uptake of siRNA in the doxorubicin-resistant human ova- rian cancer cell lines SKOV3/A were found in RGD-Lipo-siRNA preparation as compared to that of unmodified cationic lipsomes （Lipo-siRNA）. Using the post-insertion method for RGD modification, lysosome release of siRNA in pRGD-Lipo-siRNA was improved. From flow cytometry, significant increase of doxorubicin accumulation was observed in the SKOV3/A cells treated with pRGD-Lipo-siRNA targeting human MDR1 gene. In vitro cytotoxicity assay showed that the significant cell growth inhibition was achieved in the SKOV3/A cells after treating with the combined use of siRNA and doxorubicin. In conclusions, postinserted RGD modified lipoplex, pRGD-Lipo-siRNA, was successfully used for siRNA transfection and achieved drug resistance reversal in human ovarian cancer SKOV3/A （doxorubicin-resistant） cells. It suggested that this liposomes might be a potential vehicle for siRNA delivery in vivo.

Study on Liposomes of Mimetic Red Cell

Liposomes were prepared by adding hydrophilic agents PEG PE, rigidity agent SM in the bilayer membrane for mimetic red cell membrane. In PBS or serum, release of calcein content from liposomes dramatically decreased, which demonstrated increasing membrane stability by adding PEG PE or SM. The ratio b/R of the remains of liposomes in blood to that in RES was used as a parameter of biodistribution in vivo. At 2 h after iv injection, b/R of modified liposomes was enhanced 6.5～13.1 fold. Their clearance half life from blood circulation was delayed 1.6～5.8 fold. The modification of liposome membrane by PEG PE or SM is the favorable condition for drug liposomes to target the non RES.

Current developments in drug delivery with thermosensitive liposomes

Thermosensitive liposomes(TSLs) have been an important research area in the field of tumor targeted chemotherapy. Since the first TSLs appeared that using 1,2-dipalmitoyl-snglyce-ro-3-phosphocholine(DPPC) as the primary liposomal lipid, many studies have been done using this type of liposome from basic and practical aspects. While TSLs composed of DPPC enhance the cargo release near the phase transition temperature, it has been shown that many factors affect their temperature sensitivity. Thus numerous attempts have been undertaken to develop new TSLs for improving their thermal response performance. The main objective of this review is to introduce the development and recent update of innovative TSLs formulations, including combination of radiofrequency ablation(RFA), highintensity focused ultrasound(HIFU), magnetic resonance imaging(MRI) and alternating magnetic field(AMF). In addition, various factors affecting the design of TSLs, such as lipid composition, surfactant, size and serum components are also discussed.

Arsenic trioxide encapsulated liposomes prepared via copper acetate gradient loading method and its antitumor efficiency

In this study, arsenic trioxide(ATO) was encapsulated in liposomes via copper acetate(Cu(OAc)2) gradients and high entrapment efficiency of over 80% was obtained. The average particle size and the zeta-potential of the liposomes were detected to be 115.1 ± 29.1 nm and-21.97 ± 0.6 m V, respectively. The TEM images showed rod-like precipitates in the inner aqueous phase, which was supposed be due to the formation of insoluble ATO–Cu complex.The in vitro drug release of ATO–Cu liposomes exhibited a sustained release over 72 h, and the release rates decreased with the increase of the p H of release media. Pharmacokinetic and tissue distribution studies of ATO liposomes showed significantly reduced plasma clearance rate, increased AUC0–12h and T1/2, and improved tumor distribution of As compared to iv administration of ATO solution. The anti-tumor effect of ATO loaded liposomes to S180 tumor-bearing mice was significantly improved with a tumor inhibition rate of 61.2%,meanwhile the toxicity of encapsulated ATO was greatly decreased. In conclusion, ATO can be effectively encapsulated into liposomes by remote loading method via Cu(OAc)2 gradients;the co-administration of ATO and Cu(Ⅱ) via liposomal formulation may find wide applications in the treatment of various tumors.

Application of cationic liposomes for delivery of nucleic acids

Nucleic acid-based bioactive substances have recently emerged as a new class of nextgeneration therapeutics, but their development has been limited by their relatively weakdelivery into target cells. Cationic liposomes have been studied as a means to enhance thestability of nucleic acid therapeutics in the bloodstream and improve their cellular delivery.As nucleic acid therapeutics, siRNA and plasmid DNA have been extensively tested fordelivery using cationic liposomes. This review discusses recent progress in the applicationof cationic liposomes for the delivery of nucleic acid therapeutics.

Analytical methods for investigating in vivo fate of nanoliposomes:A review

Nanoliposomes are considered to be the most successful nanoparticle drug delivery system, but their fate in vivo has not been fully understood due to lack of reliable bioanalytical methods, which seriously limits the development of liposomal drugs. Hence, an overview of currently used bioanalytical methods is imperative to lay the groundwork for the need of developing a bioanalytical method for liposome measurements in vivo. Currently, major analytical methods for nanoliposomes measurement in vivo include fluorescence labeling, radiolabeling, magnetic resonance imaging（MRI）, mass spectrometry and computed tomography. In this review, these bioanalytical methods are summarized, and the advantages and disadvantages of each are discussed. We provide insights into the applicability and limitations of these analytical methods in the application of nanoliposomes measurement in vivo, and highlight the recent development of instrumental analysis techniques. The review is devoted to providing a comprehensive overview of the investigation of nanoliposomes design and associated fate in vivo, promoting the development of bioanalytical techniques for nanoliposomes measurement, and understanding the pharmacokinetic behavior, effectiveness and potential toxicity of nanoliposomes in vivo.

Formation strategies,mechanism of intracellular delivery and potential clinical applications of pH-sensitive liposomes

pH-sensitive liposomes are designed to specifically triggered release the loaded drugs in response to the change of pH in the surrounding serum.So pH-sensitive liposomes can effectively deliver drug or gene fragments into the cytoplasm via the endocytotic pathway.Furthermore,pH-sensitive liposomes can be successfully used in clinical if they enable the encapsulated drugs to be targeted to pathological tissues(such as primary tumors,metastases,local ischemia,inflammation and infection)of the body in which pH is less than the normal physiological value.That’s the reason why a growing amount of literatures described the development and applications of pH-sensitive liposomes to improve the therapeutic index of the encapsulated active ingredients.In this review,the commonly used pH-sensitive molecules for pH-sensitive liposome and the mechanisms of intracellular delivery of pH-sensitive liposomes were addressed.Besides,the potential clinical applications were fully discussed in detail with an expectation to contribute to the clinical research of pH-sensitive liposomes.