Rui Kuai

Comparison of four different peptides to enhance accumulation of liposomes into the brain

The cell penetrating peptide TAT, which appears to enter cells with alacrity, can pass through the BBB efficiently. It has been indentified to enhance the brain delivery of the liposome. However, little was known about its mechanism. TAT contains a basic region consisting of six arginine and two lysine residues. These eight basic amino acids seem to be the key to its highly efficient membrane translocation and brain delivery. In this study, four selected peptides are synthesized. (1) TAT peptide with terminal Cysteine (Cys-AYGRKKRRQRRR). (2) TAT peptide with disordered sequence (Cys-RKARYRGRKRQR). (3) Glycine and glutamic acid substituted TAT peptide (Cys-AYGGQQGGQGGG). (4) R8 (Cys-RRRRRRRR). Liposomes were chosen as the delivery vehicle. The peptide was covalently bonded with the liposome. We compare four peptides for their brain targeting potential, and investigate their ability to target liposomes to the brain in vitro and in vivo. The cellular uptake of these four liposomes by brain capillary endothelial cells (BCECs) of rats and C6s and the mechanism of the pathway of endocytosis were explored. Biodistribution in vivo was also investigated qualitatively and quantitatively. The results showed that the charge of the peptide played an important role in enhancing its brain delivery. The sequence had little to do with its membrane translocation and brain delivery indicated there might be no specific receptor or transporter for the Tat peptide.

Designer vaccine nanodiscs for personalized cancer immunotherapy

Despite the tremendous potential of peptide-based cancer vaccines, their efficacy has been limited in humans. Recent innovations in tumor exome sequencing have signaled the new era of personalized immunotherapy with patient-specific neo-antigens, but a general methodology for stimulating strong CD8α+ cytotoxic T-lymphocyte (CTL) responses remains lacking. Here we demonstrate that high density lipoprotein-mimicking nanodiscs coupled with antigen (Ag) peptides and adjuvants can markedly improve Ag/adjuvant co-delivery to lymphoid organs and sustain Ag presentation on dendritic cells. Strikingly, nanodiscs elicited up to 47-fold greater frequencies of neoantigen-specific CTLs than soluble vaccines and even 31-fold greater than perhaps the strongest adjuvant in clinical trials (i.e. CpG in Montanide). Moreover, multi-epitope vaccination generated broad-spectrum T-cell responses that potently inhibited tumor growth. Nanodiscs eliminated established MC-38 and B16F10 tumors when combined with anti-PD-1 and anti-CTLA-4 therapy. These findings represent a new powerful approach for cancer immunotherapy and suggest a general strategy for personalized nanomedicine.

Liposome formulated with TAT-modified cholesterol for improving brain delivery and therapeutic efficacy on brain glioma in animals

The treatment of central nervous system diseases such as brain glioma is a major challenge due to the presence of the blood-brain barrier (BBB). A cell-penetrating peptide TAT (AYGRKKRRQRRR), which appears to enter cells with alacrity, was employed to enhance the delivery efficiency of normal drug formulation to the brain. Targeting liposomal formulations often apply modified phospholipids as anchors. However, cholesterol, another liposomal component more stable and cheaper, has not been fully investigated as an alternative anchor. In our study, TAT was covalently conjugated with cholesterol for preparing doxorubicin-loaded liposome for brain glioma therapy. Cellular uptake by brain capillary endothelial cells (BCECs) and C6 glioma cells was explored. The anti-proliferative activity against C6s confirmed strong inhibitory effect of the liposomes modified with doxorubicin-loaded TAT. The bio-distribution findings in brains and hearts were evident of higher efficiency of brain delivery and lower cardiotoxic risk. The results on survival of the brain glioma-bearing animals indicate that survival time of the glioma-bearing rats treated with TAT-modified liposome was much longer than in the other groups. In conclusion, the potency of the TAT-modified liposome to enter the BBB appears to be related with the TAT on the liposomes surface. The TAT-modified liposome may improve the therapeutic efficacy on brain glioma in vitro and in vivo.

Liposome formulated with TAT-modified cholesterol for enhancing the brain delivery.

Delivery of drugs to the brain is a major challenge due to the presence of the blood-brain barrier (BBB). The cell penetrating peptide TAT, which appears to enter cells with alacrity, can pass through the BBB efficiently. With this in mind, a novel TAT-modified liposome (TAT-LIP) was developed for overcoming the ineffective delivery of normal drug formulation to the brain. Targeting liposomal formulations are always composed of modified phospholipids as an anchor. However, cholesterol, another liposomal component, which was more stable and cheaper, has not been fully investigated as an alternative anchor. In this study, TAT was covalently conjugated with the cholesterol to prepare the liposome. The cellular uptake by brain capillary endothelial cells (BCECs) of rats and the mechanism of TAT-LIP pathway of endocytosis was explored. The blood brain barrier model in vitro was established to evaluate the transendothelial ability crossing the BBB and its transport mechanism. The biodistribution of each formulation was further identified. The results showed that the positive charge of the TAT-LIP played an important role in enhancing its brain delivery. The absorptive endocytosis might be one of the mechanisms of TAT-LIP crossing the BBB. In conclusion, the experimental data in vitro and in vivo indicated that the TAT-LIP was a promising brain drug delivery system due to its high delivery efficiency across the BBB.

Targeted delivery of cargoes into a murine solid tumor by a cell-penetrating peptide and cleavable poly(ethylene glycol) comodified liposomal delivery system via systemic administration.

A liposomal delivery system with a high efficiency of accumulation in tumor tissue and then transportation of the cargo into tumor cells was developed here and evaluated via systemic administration. 1,2-Distearoyl-sn-glycero-3-phosphoethanolamine-poly(ethylene glycol)(2000) (DSPE-PEG(2000))-TAT and protective DSPE-PEG(2000) modified liposomes possessing good stability in 50% FBS (fetal bovine serum) and good uptake efficiency were used as the basic formulation (TAT-SL; SL = stealth liposome), and then longer cysteine (Cys)-cleavable PEG(5000) was incorporated to modulate the function of TAT. All of the formulations to be used in vivo had sizes in a range of 80-100 nm and were stable in the presence of 50% FBS. Optical imaging showed that the incorporation of cleavable PEG(5000) into TAT-SL (i.e., C-TAT-SL) led to much more tumor accumulation and much less liver distribution compared with TAT-SL. The in vivo delivery profiles of C-TAT-SL were investigated using DiD as a fluorescent probe. Confocal laser scanning microscopy and flow cytometry showed that C-TAT-SL had a 48% higher (p < 0.001) delivery efficiency in the absence of Cys and a 130% higher (p < 0.001) delivery efficiency in the presence of Cys than the control (SL), indicating the successful targeted delivery of cargo was achieved by C-TAT-SL via systemic administration especially with a subsequent administration of Cys.

In vitro and in vivo investigation of glucose-mediated brain-targeting liposomes

New glycosyl derivative of cholesterol was synthesized as a material for preparing novel liposome to overcome the ineffective delivery of normal drug formulations to brain by targeting the (glucose transporters) GLUTs on the BBB. Coumarin-6 was used as fluorescent probe. The results have shown that the cytotoxicity for the brain capillary endothelial cells (BCECs) of the glucose-mediated brain targeting liposome containing coumarin-6 was less than that of conventional liposome. The BBB model in vitro was established by coculturing of BCECs and astrocytes (ACs) of rat to test the transendothelial ability crossing the BBB. The transendothelial ability was confirmed strengthen alone with the amount of the new glycosyl derivative of cholesterol used in liposome. After i.v. administration of LIP, control liposome (CLP), and GLP-4, the AUC0–t of coumarin-6 for GLP-4 was 2.85 times higher than that of LIP, and 3.33 times higher than that of CLP. The Cmax of CLP-4 was 1.43 times higher than that of LIP, and 3.10 times higher than that of CLP. Both pharmacokinetics and distribution in mice were also investigated to show that this novel brain targeting drug delivery system was promising.

Synthetic high-density lipoprotein nanoparticles: A novel therapeutic strategy for adrenocortical carcinomas

BACKGROUND Chemotherapeutic strategies for adrenocortical carcinoma (ACC) carry substantial toxicities. Cholesterol is critical for ACC cell growth and steroidogenesis, and ACC cells overexpress scavenger receptor BI, which uptakes cholesterol from circulating high-density lipoprotein (HDL) cholesterol. We hypothesize that cholesterol-free synthetic-HDL nanoparticles (sHDL) will deplete cholesterol and synergize with chemotherapeutics to achieve enhanced anticancer effects at lesser (less toxic) drug levels. METHODS The antiproliferative efficacy of ACC cells for the combinations of sHDL with chemotherapeutics was tested by Cell-Titer Glo. Cortisol levels were measured from the culture media. Effects on steroidogenesis was measured by real-time polymerase chain reaction (RT-PCR). Induction of apoptosis was evaluated by flow cytometry. RESULTS Combination Index (CI) for sHDL and either etoposide (E), cisplatin (P), or mitotane (M) demonstrated synergy (CI < 1) for antiproliferation. Alone or in combination with the chemotherapy drugs, sHDL was able to decrease cortisol production by 70-90% compared with P alone or controls (P < .01). RT-PCR indicated inhibition of steroidogenic enzymes for sHDL (P < .01 vs no sHDL). Combination therapy with sHDL increased apoptosis by 30-50% compared with drug or sHDL alone (P < .03), confirmed by a decrease in the mitochondrial potential. CONCLUSION sHDL can act synergistically and lessen the amount of M/E/P needed for anticancer efficacy in ACC in part owing to cholesterol starvation. This novel treatment strategy warrants further investigation translationally.

Chemo-photothermal therapy combination elicits anti-tumor immunity against advanced metastatic cancer

Photothermal therapy (PTT) is a promising cancer treatment modality, but PTT generally requires direct access to the source of light irradiation, thus precluding its utility against disseminated, metastatic tumors. Here, we demonstrate that PTT combined with chemotherapy can trigger potent anti-tumor immunity against disseminated tumors. Specifically, we have developed polydopamine-coated spiky gold nanoparticles as a new photothermal agent with extensive photothermal stability and efficiency. Strikingly, a single round of PTT combined with a sub-therapeutic dose of doxorubicin can elicit robust anti-tumor immune responses and eliminate local as well as untreated, distant tumors in >85% of animals bearing CT26 colon carcinoma. We also demonstrate their therapeutic efficacy against TC-1 submucosa-lung metastasis, a highly aggressive model for advanced head and neck squamous cell carcinoma (HNSCC). Our study sheds new light on a previously unrecognized, immunological facet of chemo-photothermal therapy and may lead to new therapeutic strategies against advanced cancer.Photothermal therapy (PTT) for cancer treatment is currently limited to local, accessible, tumors. Here the authors show that PTT combination with chemotherapy, by stimulating an immune response, is effective against distant tumors and establishes immune memory, thus providing a strategy to target metastatic disease.

Effect of size and pegylation of liposomes and peptide-based synthetic lipoproteins on tumor targeting

Synthetic high-density lipoprotein nanoparticles (sHDL) are a valuable class of nanomedicines with established animal safety profile, clinical tolerability and therapeutic efficacy for cardiovascular applications. In this study we examined how the scavenger receptor B-I-mediated (SR-BI) tumor-targeting ability of sHDL, long plasma circulation half-life, and small particle size (9.6±0.2nm) impacted sHDL accumulation in SR-BI positive colorectal carcinoma cells, 3D tumor spheroids, and in vivo xenografts. We compared tumor accumulation of sHDL with that of liposomes (LIP, 130.7±0.8nm), pegylated liposomes (PEG-LIP, 101±2nm), and pegylated sHDL (12.1±0.1nm), all prepared with the same lipid components. sHDL penetrated deep (210μm) into tumor spheroids and exhibited 12- and 3-fold higher in vivo solid tumor accumulation, compared with LIP (p<0.01) and PEG-LIP (p<0.05), respectively. These results suggest that sHDL with established human safety possess promising intrinsic tumor-targeted properties.

Lupus high-density lipoprotein induces proinflammatory responses in macrophages by binding lectin-like oxidised low-density lipoprotein receptor 1 and failing to promote activating transcription factor 3 activity

Objectives Recent evidence indicates that high-density lipoprotein (HDL) exerts vasculoprotective activities by promoting activating transcription factor 3 (ATF3), leading to downregulation of toll-like receptor (TLR)-induced inflammatory responses. Systemic lupus erythematosus (SLE) is associated with increased cardiovascular disease risk not explained by the Framingham risk score. Recent studies have indicated oxidised HDL as a possible contributor. We investigated the potential mechanisms by which lupus HDL may lose its anti-inflammatory effects and promote immune dysregulation. Methods Control macrophages were challenged with control and SLE HDL in vitro and examined for inflammatory markers by real-time qRT-PCR, confocal microscopy, ELISA and flow cytometry. Lupus-prone mice were treated with an HDL mimetic (ETC-642) in vivo and inflammatory cytokine levels measured by real-time qRT-PCR and ELISA. Results Compared with control HDL, SLE HDL activates NFκB, promotes inflammatory cytokine production and fails to block TLR-induced inflammation in control macrophages. This failure of lupus HDL to block inflammatory responses is due to an impaired ability to promote ATF3 synthesis and nuclear translocation. This inflammation is dependent on lectin-like oxidised low-density lipoprotein receptor 1 (LOX1R) binding and rho-associated, coiled-coil containing protein kinase 1 and 2 (ROCK1/2) kinase activity. HDL mimetic-treated lupus mice showed significant ATF3 induction and proinflammatory cytokine abrogation. Conclusions Lupus HDL promotes proinflammatory responses through NFκB activation and decreased ATF3 synthesis and activity in an LOX1R-dependent and ROCK1/2-dependent manner. HDL mimetics should be explored as potential therapies for inflammation and SLE cardiovascular risk.