Sialic Acid Conjugate-Modified Cationic Liposomal Paclitaxel for Targeted Therapy of Lung Metastasis in Breast Cancer: What a Difference the Cation Content Makes.

Cationic lipids play a pivotal role in developing novel drug delivery systems for diverse biomedical applications, owing to the success of mRNA vaccines against COVID-19 and the Phase III antitumor agent EndoTAG-1. However, the therapeutic potential of these positively charged liposomes is limited by dose-dependent toxicity. While an increased content of cationic lipids in the formulation can enhance the uptake and cytotoxicity toward tumor-associated cells, it is crucial to balance these advantages with the associated toxic side effects. In this work, we synthesized the cationic lipid HC-Y-2 and incorporated it into sialic acid (SA)-modified cationic liposomes loaded with paclitaxel to target tumor-associated immune cells efficiently. The SA-modified cationic liposomes exhibited enhanced binding affinity toward both RAW264.7 cells and 4T1 tumor cells in vitro due to the increased ratios of cationic HC-Y-2 content while effectively inhibiting 4T1 cell lung metastasis in vivo. By leveraging electrostatic forces and ligand-receptor interactions, the SA-modified cationic liposomes specifically target malignant tumor-associated immune cells such as tumor-associated macrophages (TAMs), reduce the proportion of cationic lipids in the formulation, and achieve dual objectives: high cellular uptake and potent antitumor efficacy. These findings highlight the potential advantages of this innovative approach utilizing cationic liposomes.

Sialic acid-modified doxorubicin liposomes target tumor-related immune cells to relieve multiple inhibitions of CD8+ T cells.

In different types of cancer treatments, cancer-specific T cells are required for effective anticancer immunity, which has a central role in cancer immunotherapy. However, due to the multiple inhibitions of CD8+ T cells by tumor-related immune cells, CD8+ T-cell mediated antitumor immunotherapy has not achieved breakthrough progress in the treatment of solid tumors. Receptors for sialic acid (SA) are highly expressed in tumor-associated immune cells, so SA-modified nanoparticles are a drug delivery nanoplatform using tumor-associated immune cells as vehicles. To relieve the multiple inhibitions of CD8+ T cells by tumor-associated immune cells, we prepared SA-modified doxorubicin liposomes (SL-DOX, Scheme 1A). In our study, free SA decreased the toxicity of SL-DOX to tumor-associated immune cells. Compared with common liposomes, SL-DOX could inhibit tumor growth more effectively. It is worth noting that SL-DOX could not only kill tumor-related neutrophils and monocytes to relieve the multiple inhibitions of CD8+ T cells but also induce immunogenic death of tumor cells to promote the infiltration and differentiation of CD8+ T cells (Scheme 1B). Therefore, SL-DOX has potential value for the clinical therapeutic effect of CD8+ T cells mediating anti-tumor immunotherapy.

Sialic acid-modified docetaxel cationic liposomes: double targeting of tumor-associated macrophages and tumor endothelial cells.

Taxane drugs are clinically used for the treatment of many types of cancers due to their excellent antitumor effects. However, the surfactants contained in the injections currently used in the clinic may have serious toxic side effects on the organism, making it necessary to develop new dosage forms. Cationic liposomes have been widely used in antitumor research because of their advantage of preferentially targeting tumor neovascularization, but antitumor by targeting tumor vasculature alone does not necessarily provide good results. Malignant tumors represent complex ecosystems, tumor-associated macrophages (TAMs) and tumor endothelial cells (TECs) in the tumor microenvironment play crucial roles in tumor growth. Therefore, given the ability to achieve active targeting of TAMs and TECs by using sialic acid (SA) as a targeting material, the potential of cationic nanoformulations to preferentially target neovascularization at the tumor site, and the excellent antitumor effects of the taxane drugs docetaxel (DOC), in the present study, sialic acid-cholesterol coupling (SA-CH) was selected as a targeting material to prepare a DOC cationic liposome (DOC-SAL) for tumor therapy. The results of the study showed that DOC-SAL had the strongest drug accumulation in tumor tissues compared with the common DOC formulations, and was able to effectively reduce the colonization of TAMs, inhibit the proliferation of tumor cells, and have the best tumor-suppressing effect. In addition, DOC-SAL was able to improve the internal microenvironment of tumors by modulating cytokines. In summary, this drug delivery system has good anti-tumor effects and provides a new option for tumor therapy.

Phosphatidylserine and/or Sialic Acid Modified Liposomes Increase Uptake by Tumor-associated Macrophages and Enhance the Anti-tumor Effect.

DOX liposomes have better therapeutic effects and lower toxic side effects. The targeting ability of liposomes is one of the key factors affecting the therapeutic effect of DOX liposomes. This study developed two types of targeted liposomes. Sialic acid (SA)-modified liposomes were designed to target the highly expressed Siglec-1 receptor on tumor-associated macrophages surface. Phosphatidylserine (PS)-modified liposomes were designed to promote phagocytosis by monocyte-derived macrophages through PS apoptotic signaling. In order to assess and compare the therapeutic potential of different targeted pathways in the context of anti-tumor treatment, we compared four phosphatidylserine membrane materials (DOPS, DSPS, DPPS and DMPS) and found that liposomes prepared using DOPS as material could significantly improve the uptake ability of RAW264.7 cells for DOX liposomes. On this basis, normal DOX liposomes (CL-DOX) and SA-modified DOX liposomes (SAL-DOX), PS-modified DOX liposomes (PS-CL-DOX), SA and PS co-modified DOX liposomes (PS-SAL-DOX) were prepared. The anti-tumor cells function of each liposome on S180 and RAW264.7 in vitro was investigated, and it was found that SA on the surface of liposomes can increase the inhibitory effect. In vivo efficacy results exhibited that SAL-DOX and PS-CL-DOX were superior to other groups in terms of ability to inhibit tumor growth and tumor inhibition index, among which SAL-DOX had the best anti-tumor effect. Moreover, SAL-DOX group mice had high expression of IFN-γ as well as IL-12 factors, which could significantly inhibit mice tumor growth, improve the immune microenvironment of the tumor site, and have excellent targeted delivery potential.

Targeting Ibrutinib to Tumor-Infiltrating T Cells with a Sialic Acid Conjugate-Modified Phospholipid Complex for Improved Tumor Immunotherapy.

Immune checkpoint blockade (ICB) treatment for the clinical therapy of numerous malignancies has attracted widespread attention in recent years. Despite being a promising treatment option, developing complementary strategies to enhance the proportion of patients benefiting from ICB therapy remains a formidable challenge because of the complexity of the tumor microenvironment. Ibrutinib (IBR), a covalent inhibitor of Bruton's tyrosine kinase (BTK), has been approved as a clinical therapy for numerous B-cell malignancies. IBR also irreversibly inhibits interleukin-2 inducible T cell kinase (ITK), an essential enzyme in Th2-polarized T cells that participates in tumor immunosuppression. Ablation of ITK by IBR can elicit Th1-dominant antitumor immune responses and potentially enhance the efficacy of ICB therapy in solid tumors. However, its poor solubility and rapid clearance in vivo restrict T cell targetability and tumor accumulation by IBR. A sialic acid derivative-modified nanocomplex (SA-GA-OCT@PC) has been reported to improve the efficacy of IBR-mediated combination immunotherapy in solid tumors. In vitro and in vivo experiments showed that SA-GA-OCT@PC effectively accumulated in tumor-infiltrating T cells mediated by Siglec-E and induced Th1-dominant antitumor immune responses. SA-GA-OCT@PC-mediated combination therapy with PD-L1 blockade agents dramatically suppressed tumor growth and inhibited tumor relapse in B16F10 melanoma mouse models. Overall, the combination of the SA-modified nanocomplex platform and PD-L1 blockade offers a treatment opportunity for IBR in solid tumors, providing novel insights for tumor immunotherapy.

Liposomal Doxorubicin: the Sphingomyelin/Cholesterol System Significantly Enhances the Antitumor Efficacy of Doxorubicin.

Doxorubicin (DOX) has a cytotoxic effect on many tumor cells; however, its clinical application is limited owing to its strong side effects. Although Doxil® reduces the cardiotoxicity of free DOX, it has also introduced a new dose-limiting toxicity. In a previous study, a sialic acid-cholesterol conjugate (SA-CH) was synthesized and modified onto the surface of DOX-loaded liposomes to target tumor-associated macrophages (TAMs), further improving the efficacy of DOX-loaded liposomes over that of Doxil®. Meanwhile, the good retention characteristics and promising antitumor ability of sphingomyelin/cholesterol (SM/CH) system for water-soluble drugs have attracted wide attention. Therefore, we aimed to use SA-CH as the target and hydrogenated soybean phosphatidylcholine (HSPC) or egg sphingomyelin (ESM) as the membrane material to develop a more stable DOX-loaded liposome with stronger antitumor activity. The liposomes were evaluated for particle size, polydispersity index, zeta potential, entrapment efficiency, in vitro release, long-term storage, cytotoxicity, cellular uptake, pharmacokinetics, tumor targetability, and in vivo antitumor activity. In the liposomes prepared using HSPC/CH, sialic acid (SA) modification considerably increased the accumulation of DOX-loaded liposomes in the tumor, thus exerting a better antitumor effect. However, SA modification in DOX-ESL (SA-CH-modified DOX-loaded liposomes prepared by ESM/CH) destroyed the strong retention effect of the ESM/CH system on DOX, resulting in a reduced antitumor effect. Notably, DOX-ECL (DOX-loaded liposome prepared by ESM/CH) had the optimal storage stability, lowest toxicity, and optimal antitumor effect due to better drug retention properties. Thus, the ESM/CH liposome of DOX is a potential drug delivery system. Sketch of the effect of two DOX-loaded liposomes with hydrogenated soybean phospholipid (HSPC) and egg sphingomyelin (ESM) as lipid membrane material and surface-modified SA derivative on tumor growth inhibition.

Polysialic Acid Self-assembled Nanocomplexes for Neutrophil-Based Immunotherapy to Suppress Lung Metastasis of Breast Cancer.

The role of neutrophils in tumor metastasis has recently attracted widespread interest. Neutrophils are the most abundant immune cells in human peripheral blood, and large numbers can spontaneously migrate to metastatic sites, where they form an immunosuppressive microenvironment. Polysialic acid (PSA) can target peripheral blood neutrophils (PBNs) mediated by L-selectin, and abemaciclib (ABE) and mitoxantrone (MIT) can treat immunosuppressive microenvironments. Here, we aimed to inhibit lung metastasis of breast cancer and improve chemoimmunotherapy by designing a PSA-modified ABE and MIT co-delivery system (AM-polyion complex (PIC)) to target PBNs in mice with metastatic tumors. We found that through electrostatic interactions between the strong negative charge of PSA and the positive charge of the drug can form stable nanocomplexes and that spontaneous migration of neutrophils can mediate the aggregation of these complexes in the lungs, induce antimetastatic immune responses, enhance the effectiveness of cytotoxic T lymphocytes (CTLs), and inhibit regulatory T cell (Treg) proliferation in vivo and in vitro. Pharmacodynamic results suggested that neutrophil-mediated AM-PIC chemoimmunotherapy inhibited tumor metastasis in mice with lung metastasis of 4T1 breast cancer. Overall, PSA-modified nanocomplexes offer promising neutrophil-mediated, targeted drug delivery systems to treat lung metastasis of breast cancer.

An Application of Tumor-Associated Macrophages as Immunotherapy Targets: Sialic Acid-Modified EPI-Loaded Liposomes Inhibit Breast Cancer Metastasis.

Breast cancer metastasis is an important cause of death in patients with breast cancer and is closely related to circulating tumor cells (CTCs) and the metastatic microenvironment. As the most infiltrating immune cells in the tumor microenvironment (TME), tumor-associated macrophages (TAMs), which highly express sialic acid (SA) receptor (Siglec-1), are closely linked to tumor progression and metastasis. Furthermore, the surface of CTCs also highly expressed receptor (Selectin) for SA. A targeting ligand (SA-CH), composed of SA and cholesterol, was synthesized and modified on the surface of epirubicin (EPI)-loaded liposomes (EPI-SL) as an effective targeting delivery system. Liposomes were evaluated for characteristics, stability, in vitro release, cytotoxicity, cellular uptake, pharmacokinetics, tumor targeting, and pharmacodynamics. In vivo and in vitro experiments showed that EPI-SL enhanced EPI uptake by TAMs. In addition, cellular experiments showed that EPI-SL could also enhance the uptake of EPI by 4T1 cells, resulting in cytotoxicity second only to that of EPI solution. Pharmacodynamic experiments have shown that EPI-SL has optimal tumor inhibition with minimal toxicity, which can be ascribed to the fact that EPI-SL can deliver drugs to tumor based on TAMs and regulate TME through the depletion of TAMs. Our study demonstrated the significant potential of SA-modified liposomes in antitumor metastasis. Schematic diagram of the role of SA-CH modified EPI-loaded liposomes in the model of breast cancer metastasis.

Antitumor Immunotherapy of Sialic Acid and/or GM1 Modified Coenzyme Q10 Submicron Emulsion.

Immunotherapy is a novel therapeutic approach for controlling and killing tumor cells by stimulating or reconstituting the immune system, among which T cells serve as immune targets. Herein, we used coenzyme Q10 (CoQ10), which has both immune activation and avoids adverse reactions, as a model drug and developed four CoQ10 submicron emulsions modified with sialic acid (SA) and/or monosialotetrahexosyl ganglioside (GM1). On the one hand, SA interacts with L-selectins on the surface of T cells after entering the circulatory system, leading to activation of T cells and enhancement of antitumor immune responses. On the other hand, owing to its immune camouflage, GM1 can prolong the circulation time of the preparation in the body, thereby increasing the accumulation of the drug at the tumor site. In vitro and in vivo experiments showed that SA-modified preparations exhibited stronger immune activation and inhibition of tumor proliferation. Pharmacokinetic experiments showed that GM1-modified preparations have longer circulation times in vivo. However, SA and GM1 co-modification did not produce a synergistic effect on the preparation. In conclusion, the SA-modified CoQ10 submicron emulsion (Q10-SE) showed optimal antitumor efficacy when administered at a medium dose (6 mg CoQ10 kg-1). In this study, the submicron emulsion model was used as a carrier, and the tumor-bearing mice were used as animal models. In addition, CoQ10 submicron emulsion was modified with SA-CH with active targeting function and/or GM1 with long-circulation function to explore the antitumor effects of different doses of CoQ10 submicron emulsion, and to screen the best tumor immunotherapy formulations of CoQ10.

Evaluation of the Antitumor Effect and Immune Response of Micelles Modified with a Polysialic Acid-D-α-Tocopheryl Polyethylene Glycol 1000 Succinate Conjugate.

D-α-Tocopheryl polyethylene glycol 1000 succinate (TPGS) has shown potential applications in cancer therapy owing to its attractive properties, including reversal of multi-drug resistance and synergistic effects with antitumor drugs. However, its associated shortcomings cannot be underestimated, including activation of the body's immune response and acceleration of blood clearance of polyethylene glycolylated preparations. Polysialic acid (PSA) is a polysaccharide homopolymer, with the dual function of immune camouflage and tumor targeting. PSA and TPGS conjugates (PSA-TPGS) were synthesized to weaken the immune risks of TPGS. We developed PSA-TPGS and TPGS self-assembled mixed micelles and encapsulated the classical antineoplastic, docetaxel. The particle size of docetaxel-loaded mixed micelles was 16.3 ± 2.0 nm, with entrapment efficiency of 99.0 ± 0.9% and drug-loading efficiency of 3.20 ± 0.03%. Antitumor activity studies revealed that the mixed micelles showed better tumor inhibition than Tween 80 and TPGS micelles. Detection of the accelerated blood clearance (ABC) phenomenon demonstrated that insertion of PSA-TPGS into the micelles weakened the ABC phenomenon induced by TPGS. In summary, PSA-TPGS could be a potential nanocarrier to improve antitumor activity and weaken immune responses.

Sialic Acid Conjugate-Modified Liposomal Dexamethasone Palmitate Targeting Neutrophils for Rheumatoid Arthritis Therapy: Influence of Particle Size.

Many anti-inflammatory therapies targeting neutrophils have been developed so far. A sialic acid (SA)-modified liposomal (SAL) formulation, based on the high expression of L-selectin in peripheral blood neutrophils (PBNs) and SA as its targeting ligand, has proved to be an effective neutrophil-mediated drug delivery system targeting rheumatoid arthritis (RA). The objective of this study was to investigate the influence of particle size of drug-carrying SALs transported and delivered by neutrophils on their anti-RA effect. Dexamethasone palmitate-loaded SALs (DP-SALs) of different particle sizes (300.2 ± 5.5 nm, 150.3 ± 4.3 nm, and 75.0 ± 3.9 nm) were prepared with DP as a model drug. Our study indicated that DP-SALs could efficiently target PBNs, with larger liposomes leading to higher drug accumulation in cells. However, a high intake of large DP-SALs by PBNs inhibited their migration ability and capacity to release the payload at the target site. In contrast, small DP-SALs (75.0 ± 3.9 nm) could maintain the drug delivery potential of PBNs, leading to their efficient accumulation at the inflammatory site, where PBNs would be excessively activated to form neutrophil extracellular traps along with efficient payload release (small DP-SALs) and finally to induce excellent anti-RA effect.

Influence of Dose on Neutrophil-Mediated Delivery of Nanoparticles for Tumor-Targeting Therapy Strategies.

It is well known that neutrophil-mediated delivery of therapeutic agents is a promising method for treating tumors. However, owing to the limited number and limited uptake ability of neutrophils, determining a reasonable dose has become an urgent problem to be solved. Furthermore, the number of nanoparticles is far greater than the number of neutrophils at normal doses, which causes excessive nanoparticles to reach nontargeted organs or tissues, leading to serious adverse effects. To address these problems, a neutrophil-targeting delivery system (DiR-DADGC-L) based on DiR-labeled and butanedioic acid (DA)-linked 5-amino-3,5-dideoxy-D-Glycerol-D-galactonanulose-cholesterol conjugate (DADGC) was designed to improve the efficiency of hitchhiking neutrophils through the specific binding of sialic acid (SA) to L-selectin (SA-binding receptor, expressed on neutrophils). DiR-DADGC-L was prepared with favorable particle size and encapsulation efficiency (%EE) to deliver DiR into neutrophils. Subsequently, diverse doses of DiR-DADGC-L were injected intravenously into S180 tumor-bearing and cyclophosphamide-depleted (CTX-D) S180 tumor-bearing mice to evaluate the in vivo behavior of liposomes. The results verified the following: a) The content of DiR-DADGC-L in neutrophils accounts for approximately 14.5% of the content of DiR-DADGC-L in plasma, and the uptake capacity of neutrophils remains unchanged under different doses, and b) both neutrophils and the enhanced permeability and retention (EPR) effect might exert significant roles in tumor treatment. As for the neutrophil-mediated delivery system, higher doses are not necessarily appropriate, and a lower dose may achieve an unexpected effect. It will be wise to determine an optimum dose to improve delivery efficiency.

Chimeric antigens receptor T cell therapy as a bridge to haematopoietic stem cell transplantation for refractory/ relapsed B-cell acute lymphomalastic leukemia.

Although chimeric antigen receptor T cells (CAR-T) targeted at CD19 or CD22 have achieved high complete remission (CR) in refractory/relapsed B-cell acute lymphoblastic leukaemia (B-ALL), it is uncertain if allogeneic haematopoietic stem cell transplantation (allo-HSCT) should be performed after CAR-T therapy to accomplish a sustainable remission. Fifty-two cases with relapsed/refractory B-ALL who underwent allo-HSCT after CR by CD19 or CD22 CAR-T were enrolled. The median time from CAR-T infusion to allo-HSCT was 50 (34-98) days. Myeloablative reduced-intensity conditioning (RIC) with total body irradiation/fludarabine-based or busulfan/fludarabine-based regimens was used. Incidences of grade II-IV acute graft-versus-host disease (aGVHD) and severe aGVHD were 23·1% and 5·8% respectively. Of 48 evaluable cases, 16 developed chronic GVHD (cGVHD) and in three of them the pattern was extensive. With a median follow-up of 334 (41-479) days, one-year overall survival and event-free survival (EFS) were 87·7% and 73·0%. One-year relapse rate and transplant-related mortality (TRM) were 24·7% and 2·2% respectively. With quick bridge to allo-HSCT after CAR-T therapy, high EFS for refractory/relapsed B-ALL has been achieved in this relatively large cohort. Our myeloablative RIC regimens have resulted in low incidences of aGVHD, cGVHD, viral reactivation and very low TRM even majority of transplants from haploidentical donors. Long-term follow-up is warranted.

Accelerated Blood Clearance of Nanoemulsions Modified with PEG-Cholesterol and PEG-Phospholipid Derivatives in Rats: The Effect of PEG-Lipid Linkages and PEG Molecular Weights.

Various types of nanocarriers modified with poly(ethylene glycol) (PEG) exhibit the accelerated blood clearance (ABC) phenomenon, resulting in reduced circulation time and abnormal increase in hepatic and splenic accumulations. Based on the abundance of esterases in the serum of rats, we developed cleavable methoxy PEG-cholesteryl methyl carbonate (mPEG-CHMC) with a carbonate linkage and noncleavable N-(carbonyl-methoxy PEG-n)-1,2-distearoyl-sn-glycero-3-phos-phoethanolamine (mPEG-DSPE) with a carbamate linkage on the surface of the nanoemulsions (CHMCE and PE, respectively). Both PEG derivatives possessed PEG with six different molecular weights (n = 350, 550, 750, 1000, 2000, and 5000). The pharmacokinetic behaviors and biodistributions of single and repeated injection of the two types of PEGylated nanoemulsions were determined to investigate the influence of cleavable linkages and PEG molecular weights on the ABC phenomenon in an attempt to find a potential strategy to eliminate the ABC phenomenon. CHMCEns (n = 1000, 2000, and 5000) exhibited the same pharmacokinetic behaviors as PE550 and PE750 and only alleviated the ABC phenomenon to a certain extent at the expense of shortened cycle time, indicating that the cleavable carbonate linkage was not an ideal strategy to eliminate the ABC phenomenon. As the molecular weights of PEG increased, the ABC phenomenon became more severe. Surprisingly, PE5000 induced a lower anti-PEG IgM level and a weaker ABC phenomenon compared with PE2000 while possessing a similar long circulation time. The results suggested that increasing the molecular weight of PEG in the PEG derivatives could be a potential strategy for eliminating the ABC phenomenon while simultaneously guaranteeing longer circulation time.

The Contribution of PEG Molecular Weights in PEGylated Emulsions to the Various Phases in the Accelerated Blood Clearance (ABC) Phenomenon in Rats.

PEGylated preparations will be cleared rapidly from blood circulation when they are administrated twice in the same animal at a time interval, referred to as the "accelerated blood clearance" (ABC) phenomenon. Commonly, the study of the ABC phenomenon was investigated in two aspects: induction phase and effectuation phase. Herein, we report the influence of physicochemical properties (PEG molecular weights) in the induction phase and effectuation phase on the ABC phenomenon. In the experiment, on one hand, PEGylated emulsions with different molecular weights of PEG (refer to PEn, n = 400, 600, 800, 1000, 2000, and 5000) were injected for the first dose (induction phase) and induced PE2000 to produce ABC phenomenon. On the other hand, after PE2000 injected, PEn was injected for the second dose (effectuation phase). The results indicated that PE2000 and PE5000 induced an intense ABC phenomenon by their long-circulating characteristic. Interestingly, PE400, PE600, PE800, and PE1000 produced a consistent ABC phenomenon but different circulation time. Apparently, the induction of the ABC phenomenon is not only determined by the circulation time but also by the PEG molecular weights. When PEn is in the effectuation phase, the extent of the ABC phenomenon was not positively related to the molecular weights of PEG, increasing first and then weaken with the increase of molecular weights of PEG. These suggest that the number of -(CH2CH2O)n- repeat units of PE2000 was more conducive to interact with anti-PEG IgM. The results reported here clearly indicate that both the PEG molecular weights of prior dose and the subsequent dose of emulsion strongly influence the extent of the ABC phenomenon. Taken together, our observations in this study complete the effect of PEG molecular weights at a different phase of the ABC phenomenon. Furthermore, our findings have a significant impact on the choice of molecular weights for PEGylated formulations for use in cross-administration.

Effects of Uncleavable and Cleavable PEG-Lipids with Different Molecular Weights on Accelerated Blood Clearance of PEGylated Emulsions in Beagle Dogs.

To investigate the effect of polyethylene glycol (PEG) molecular weights on circulation time of PEGylated emulsions and the second injection of injected PEGylated emulsions, we studied the effect of molecular weights on the pharmacokinetic behavior of PEG-DSPE (modified emulsions with 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-n-[methoxy (polyethyleneglycol)]) and PEG-CHMC (modified emulsions with poly(ethyleneglycol)-cholesteryl carbonate) emulsions in beagle dogs. The "accelerated blood clearance" (ABC) phenomenon was induced. Through this study, the contribution of PEG molecular weights on the ABC phenomenon was further clarified, and the results provided guidance for lessening or eliminating the ABC phenomenon. We injected different PEG-modified emulsions with 10% PEG-modified density into beagle dogs at 2 µmol phospholipids kg-1 and the blood samples were drawn after 1 min, 3 min, 5 min, 10 min, 15 min, 30 min, 60 min, 120 min, 240 min, 360 min, 600 min, and 24 h. Then, concentrations of the drug were assayed using high-performance liquid chromatography (HPLC). The results showed that the circulation times of PEG-DSPE-modified emulsions were significantly different because of the difference in molecular weights, whereas those of the PEG-CHMC modified emulsions were not. The spatial conformation of PEG with small molecular weights (PEG400, PEG600, and PEG800) was more likely to induce a strong ABC phenomenon. The results of our work suggest the interaction of circulation time and PEG molecular weights on the ABC phenomenon, implying that the spatial conformation of PEG has advantages that alleviate the ABC phenomenon. Importantly, the results have implications for the choice of molecular weights of PEG for PEGylated formulations.

A Sialylated-Bortezomib Prodrug Strategy Based on a Highly Expressed Selectin Target for the Treatment of Leukemia or Solid Tumors.

PURPOSE: This study aimed to explore the potential of sialic acid - related selectin targeting strategy in the treatment of leukemia and some solid tumors. We expected it could "actively" bind tumor cells and kill them, reducing non-specific toxicity to normal cells. METHODS: BOR-SA prodrug was synthesized by reacting an ortho-dihydroxy group in SA with a boronic acid group in BOR. Two kinds of leukemia cells (RAW264.7 and HL60 cells), one solid sarcoma cell model (S180 cells) and their corresponding normal cells (monocytes (MO), neutrophil (NE) and fibroblast (L929)) were selected for the in vitro cell experiments (cytotoxicity, cellular uptake, cell cycle and apoptosis experiments). The S180 tumor-bearing Kunming mice model was established for anti-tumor pharmacodynamic experiments. RESULTS: In vitro cell assay results showed that uptake of BOR-SA by HL60 and S180 cells were increased compared with the control group. BOR-SA induced a lower IC50, higher ratio of apoptosis and cell cycle arrest of tumor cells. In vivo anti-S180 tumor pharmacodynamics experiments showed that mice in the BOR-SA group had higher tumor inhibition rate, higher body weight and lower immune organ toxicity compared with the control group. CONCLUSIONS: sialic acid-mediated selectin targeting strategy may have great potential in the treatment of related tumors.

Neutrophil-Mediated Delivery of Dexamethasone Palmitate-Loaded Liposomes Decorated with a Sialic Acid Conjugate for Rheumatoid Arthritis Treatment.

PURPOSE: The aim of this research was to design dexamethasone palmitate (DP) loaded sialic acid modified liposomes, with the eventual goal of using peripheral blood neutrophils (PBNs) that carried drug-loaded liposomes to improve the therapeutic capacity for rheumatoid arthritis (RA). METHODS: A sialic acid - cholesterol conjugate (SA-CH) was synthesized and anchored on the surface of liposomal dexamethasone palmitate (DP-SAL). The physicochemical characteristics and in vitro cytotoxicity of liposomes were evaluated. Flow cytometry and confocal laser scanning microscopy were utilized to investigate the accumulation of liposomes in PBNs. The adjuvant-induced arthritis was adopted to investigate the targeting ability and anti-inflammatory effect of DP loaded liposomes. RESULTS: Both DP-CL and DP-SAL existed an average size less than 200 nm with remarkably high encapsulation efficiencies more than 90%. In vitro and in vivo experiments manifested SA-modified liposomes provided a reinforced accumulation of DP in PBNs. As well, DP-SAL displayed a greater degree of accumulation in the joints and a stronger anti-inflammatory effect in terms of RA suppression. CONCLUSIONS: SA-modified liposomal DP was a promising candidate for RA-targeting treatment through the neutrophil-mediated drug delivery system.

Dual-Ligand Modification of PEGylated Liposomes Used for Targeted Doxorubicin Delivery to Enhance Anticancer Efficacy.

Mannose receptor (CD206) and E-selectin are selectively expressed in M2-like tumor-associated macrophages (M2-TAMs) and activated endothelial cells of vessels surrounding tumor tissues. With the knowledge that D-mannose is the natural ligand of mannose receptors and L-fucose is the key calcium chelator for tumor-associated carbohydrate antigens (TACAs) binding to E-selectin, herein, we firstly reported D-mannose polyethylene glycol (PEG) conjugates (Man-PEG) and L-fucose PEG conjugates (Fuc-PEG) co-modified liposomal doxorubicin (DOX-MFPL) to improve tumor-targeting ability. The dual-ligand modified PEGylated liposomes (DOX-MFPL) were assessed by both in vitro and in vivo trials. Compared with the single-ligand D-mannose- or L-fucose-modified liposomes (DOX-MPL or DOX-FPL), DOX-MFPL achieved an increased distribution of DOX in tumor tissues. The antitumor study based on S180 tumor-bearing mice was conducted and the superior tumor inhibitory rate was shown with DOX-MFPL, probably owing to the superior tumor-targeting effect of DOX-MFPL and the modulation of the tumor microenvironment with the exhaustion of TAMs. In general, the dual-ligand drug delivery systems are expected to be promising in the development of specific and efficient methods for tumor treatment.

Enhanced Opsonization-Independent Phagocytosis and High Response Ability to Opsonized Antigen-Antibody Complexes: A New Role of Kupffer Cells in the Accelerated Blood Clearance Phenomenon upon Repeated Injection of PEGylated Emulsions.

The accelerated blood clearance (ABC) phenomenon is an immune response against the first injection of PEGylated colloidal drug delivery systems (CDDSs), which causes the accelerated clearance of the second dose. The enhanced complement-mediated phagocytic activity of Kupffer cells is responsible for accelerated second-dose clearance. Nevertheless, few studies have focused on the role of Kupffer cells in the induction phase of the ABC phenomenon. In this study, the intrinsic phagocytic activity of Kupffer cells was significantly enhanced at 6 days after the initial injected PEGylated emulsions (PEs) using the carbon clearance test and single-pass liver perfusion experiment. Furthermore, PE could stimulate Kupffer cells activation, leading to enhanced cell viability of Kupffer cells and opsonization-independent cellular uptake. It was also found that the response ability of Kupffer cells to the antigen-antibody complexes was augmented by the first injection of PE. Conclusively, we proposed that, besides anti-PEG IgM and complement activation-mediated hepatic uptake, enhanced opsonization-independent phagocytosis of Kupffer cells and the high response ability to opsonized antigen-antibody complexes contribute to the accelerated clearance of the second administration. The results indicated that Kupffer cells play an indispensable role in the ABC phenomenon and provided novel insights into the current view on the mechanism of the ABC effect.