Renal tubular epithelial cells (RTECs) are important target cells for the development of kidney-targeted drug delivery systems. Under physiological conditions, RTECs are under constant fluid shear stress (FSS) from original urine in the renal tubule and respond to changes of FSS by altering their morphology and receptor expression patterns, which may affect reabsorption and cellular uptake. Using a microfluidic system, controlled shear stress was applied to proximal tubule epithelial cell line HK-2. Next, 2-glucosamine, bovine serum albumin, and albumin nanoparticles were selected as representative carriers to perform cell uptake studies in HK-2 cells using the microfluidic platform system with controlled FSS. FSS is proven to impact the morphology of HK-2 cells and upregulate the levels of megalin and clathrin, which then led to enhanced cellular uptake efficiencies of energy-driven carrier systems such as macromolecular and albumin nanoparticles in HK-2 cells. To further investigate the effects of FSS on endocytic behavior mediated by related receptors, a mice model of acute kidney injury with reduced fluid shear stress was established. Consistent with in vitro findings, in vivo studies have also shown reduced fluid shear stress down-regulated the levels of megalin receptors, thereby reducing the renal distribution of albumin nanoparticles.

Poor distribution of antigen/adjuvant to target sites and inadequate induction of T cell responses remain major challenges in cancer immunotherapy because of the lack of appropriate delivery systems. Nanocarrier-based antigen delivery systems have emerged as an innovative strategy to improve vaccine efficacy. Here we present polymeric hybrid micelles (PHMs) as a simple and potent antigen/adjuvant co-delivery system with highly tunable properties. PHMs consist of two amphiphilic diblock copolymers, polycaprolactone-polyethylenimine (PCL-PEI) and polycaprolactone-polyethyleneglycol (PCL-PEG). PHMs with different proportions of cationic PCL-PEI were prepared and loaded with tyrosinase-related protein 2 (Trp2) peptide and adjuvant CpG oligodeoxynucleotide to generate the Trp2/PHM/CpG co-delivery system. Lymphatic and intracellular antigen delivery as a function of PCL-PEI proportion was evaluated in vitro and in vivo. PHMs containing 10% (w/w) PCL-PEI (Trp2/PHM10/CpG) showed the optimal balance of good distribution to lymph nodes, strong immunization effect after subcutaneous administration, and low toxicity to dendritic cells. In a mouse model of B16F10 melanoma, Trp2/PHM10/CpG showed significantly higher antigen-specific cytotoxic T lymphocyte activity and greater anticancer efficacy than Trp2/PHM0/CpG without PCL-PEI or a mixture of free Trp2 and CpG. These results provide new insights into how cationic segments affect the efficiency of antigen delivery by cationic nanocarriers. They also suggest that PHMs can serve as a structurally simple and highly tunable platform for co-delivery of antigen and adjuvant in cancer immunotherapy.

A phospholipid-based injectable gel was developed for the sustained delivery of leuprolide acetate (LA). The gel system was prepared using biocompatible materials (SPME), including soya phosphatidyl choline (SPC), medium chain triglyceride (MCT) and ethanol. The system displayed a sol state with low viscosity in vitro and underwent in situ gelation in vivo after subcutaneous injection. An in vitro release study was performed using a dialysis setup with different release media containing different percentages of ethanol. The stability of LA in the SPME system was investigated under different temperatures and in the presence of various antioxidants. In vivo studies in male rats were performed to elucidate the pharmacokinetic profiles and pharmacodynamic efficacy. A sustained release of LA for 28 days was observed without obvious initial burst in vivo. The pharmacodynamic study showed that once-a-month injection of LA-loaded SPME (SPME-LA) led to comparable suppression effects on the serum testosterone level as observed in LA solution except for the onset time. These findings demonstrate excellent potential for this novel SPME system as a sustained release delivery system for LA.

Facing the threat of highly variable virus infection, versatile vaccination systems are urgently needed. Intranasal mRNA vaccination provides a flexible and convenient approach. However, the nasal epithelium remains a major biological barrier to deliver antigens to nasal associated lymphoid tissue (NALT). To address this issue, a potent polymer-based intranasal mRNA vaccination system for HIV-1 treatment was synthesized using cationic cyclodextrin-polyethylenimine 2k conjugate (CP 2k) complexed with anionic mRNA encoding HIV gp120. The delivery vehicle containing CP 2k and mRNA overcame the epithelial barrier by reversibly opening the tight junctions, enhanced the paracellular delivery of mRNA and consequently minimized absorption of toxins in the nasal cavity. Together with the excellent intracellular delivery and prolonged nasal residence time, strong system and mucosal anti-HIV immune responses as well as cytokine productions were achieved with a balanced Th1/Th2/Th17 type. Our study provided the first proof of evidence that cationic polymers can be used as safe and potent intranasal mRNA vaccine carriers to overcome the nasal epithelial barrier. The safe and versatile polymeric delivery system represents a promising vaccination platform for infectious diseases.

Intelligent responsive drug delivery system opens up new avenues for realizing safer and more effective combination immunotherapy. Herein, a kind of tumor cascade-targeted responsive liposome (NLG919@Lip-pep1) is developed by conjugating polypeptide inhibitor of PD-1 signal pathway (AUNP-12), which is also a targeted peptide that conjugated with liposome carrier through matrix metalloproteinase-2 (MMP-2) cleavable peptide (GPLGVRGD). This targeted liposome is prepared through a mature preparation process, and indoleamine-2,3-dioxygenase (IDO) inhibitor NLG919 was encapsulated into it. Moreover, mediated by the enhanced permeability and retention effect (EPR effect) and AUNP-12, NLG919@Lip-pep1 first targets the cells that highly express PD-L1 in tumor tissues. At the same time, the over-expressed MMP-2 in the tumor site triggers the dissociation of AUNP-12, thus realizing the precise block of PD-1 signal pathway, and restoring the activity of T cells. The exposure of secondary targeting module II VRGDC-NLG919@Lip mediated tumor cells targeting, and further relieved the immunosuppressive microenvironment. Overall, this study offers a potentially appealing paradigm of a high efficiency, low toxicity, and simple intelligent responsive drug delivery system for targeted drug delivery in breast cancer, which can effectively rescue and activate the body's anti-tumor immune response and furthermore achieve effective treatment of metastatic breast cancer.

Macromolecular prodrugs obtained by covalently conjugating small molecular drugs with polymeric carriers were proven to accomplish controlled and sustained release of the therapeutic agents in vitro and in vivo. Polyethylene glycol (PEG) has been extensively used due to its low toxicity, low immunogenicity and high biocompatibility. However, for linear PEG macromolecules, the number of available hydroxyl groups for drug coupling does not change with the length of polymeric chain, which limits the application of PEG for drug conjugation purposes. To increase the drug loading and prolong the retention time of 5-fluorouracil (5-Fu), a macromolecular prodrug of 5-Fu, 5-fluorouracil-1 acid-PAE derivative (5-FA-PAE) was synthesized and tested for the antitumor activity in vivo.

The gene knockdown activity of small interfering RNA (siRNA) has led to their use as potential therapeutics for a variety of diseases. However, successful gene therapy requires safe and efficient delivery systems. In this study, we choose mPEG-PLGA-PLL nanoparticles (PEAL NPs) with ultrasound targeted microbubble destruction (UTMD) to efficiently deliver siRNA into cells. An emulsification-solvent evaporation method was used to prepare siRNA-loaded PEAL NPs. The NPs possessed an average size of 132.6±10.3 nm (n=5), with a uniform spherical shape, and had an encapsulation efficiency (EE) of more than 98%. As demonstrated by MTT assay, neither PEAL NPs nor siRNA-loaded PEAL NPs showed cytotoxicity even at high concentrations. The results of cellular uptake showed, with the assistance of UTMD, the siRNA-loaded PEAL NPs can be effectively internalized and can subsequently release siRNA in cells. Taken together, PEAL NPs with UTMD may be highly promising for siRNA delivery, making it possible to fully exploit the potential of siRNA-based therapeutics.

As known, the benefits of photothermal therapy (PTT) are greatly limited by the heat tolerance of cancer cells resulting from overexpressed heat shock proteins (HSPs). Then HSPs further trigger the formation of stress granules (SGs) that regulate protein expression and cell viability under various stress conditions. Inhibition of SG formation can sensitize tumor cells to PTT. Herein, we developed PEGylated pH (low) insertion peptide (PEG-pHLIP)-modified hollow copper sulfide nanoparticles (HCuS NPs) encapsulating the SG inhibitor ISRIB, with the phase-change material lauric acid (LA) as a gate-keeper, to construct a pH-driven and NIR photo-responsive controlled smart drug delivery system (IL@H-PP). The nanomedicine could specifically target slightly acidic tumor sites. Upon irradiation, IL@H-PP realized PTT, and the light-controlled release of ISRIB could effectively inhibit the formation of PTT-induced SG to sensitize tumor cells to PTT, thereby increasing the antitumor effect and inducing potent immunogenic cell death (ICD). Moreover, IL@H-PP could promote the production of reactive oxygen species (ROS) by tumor-associated macrophages (TAMs), repolarizing them towards the M1 phenotype and remodeling the immunosuppressive microenvironment. In vitro/vivo results revealed the potential of PTT combined with SG inhibitors, which provides a new paradigm for antitumor and anti-metastases.

Due to the critical correlation between inflammation and carcinogenesis, a therapeutic candidate with anti-inflammatory activity may find application in cancer therapy. Here, we report the therapeutic efficacy of celastrol as a promising candidate compound for treatment of pancreatic carcinoma via naïve neutrophil membrane-coated poly(ethylene glycol) methyl ether-block-poly(lactic-co-glycolic acid) (PEG-PLGA) nanoparticles. Neutrophil membrane-coated nanoparticles (NNPs) are well demonstrated to overcome the blood pancreas barrier to achieve pancreas-specific drug delivery in vivo. Using tumor-bearing mice xenograft model, NNPs showed selective accumulations at the tumor site following systemic administration as compared to nanoparticles without neutrophil membrane coating. In both orthotopic and ectopic tumor models, celastrol-loaded NNPs demonstrated greatly enhanced tumor inhibition which significantly prolonged the survival of tumor bearing mice and minimizing liver metastases. Overall, these results suggest that celastrol-loaded NNPs represent a viable and effective treatment option for pancreatic carcinoma.

Glioma, one of the most common brain tumors, remains a challenge worldwide. Due to the specific biological barriers such as blood-brain barrier (BBB), cancer stem cells (CSCs), tumor associated macrophages (TAMs), and vasculogenic mimicry channels (VMs), a novel versatile targeting delivery for anti-glioma is in urgent need. Here, we designed a hyaluronic acid (HA) ion-pairing nanoparticle. Then, these nanoparticles were encapsulated in liposomes, termed as DOX-HA-LPs, which showed near-spherical morphology with an average size of 155.8 nm and uniform distribution (PDI = 0.155). HA was proven to specifically bind to CD44 receptor, which is over-expressed on the surface of tumor cells, other associated cells (such as CSCs and TAMs) and VMs. We systematically investigated anti-glioma efficacy and mechanisms in vivo and in vitro. The strong anti-glioma efficacy could attribute to the accumulation in glioma site and the regulation of tumor microenvironment with depletion of TAMs, inhibition of VMs, and elimination of CSCs.

Acute liver injury (ALI) has an elevated fatality rate due to untimely and ineffective treatment. Although, schisandrin B (SchB) has been extensively used to treat diverse liver diseases, its therapeutic efficacy on ALI was limited due to its high hydrophobicity. Palmitic acid-modified serum albumin (PSA) is not only an effective carrier for hydrophobic drugs, but also has a superb targeting effect via scavenger receptor-A (SR-A) on the M1 macrophages, which are potential therapeutic targets for ALI. Compared with the common macrophage-targeted delivery systems, PSA enables site-specific drug delivery to reduce off-target toxicity. Herein, we prepared SchB-PSA nanoparticles and further assessed their therapeutic effect on ALI. In vitro, compared with human serum albumin encapsulated SchB nanoparticles (SchB-HSA NPs), the SchB-PSA NPs exhibited more potent cytotoxicity on lipopolysaccharide (LPS) stimulated Raw264.7 (LAR) cells, and LAR cells took up PSA NPs 8.79 times more than HSA NPs. As expected, the PSA NPs also accumulated more in the liver. Moreover, SchB-PSA NPs dramatically reduced the activation of NF-κB signaling, and significantly relieved inflammatory response and hepatic necrosis. Notably, the high dose of SchB-PSA NPs improved the survival rate in 72 h of ALI mice to 75%. Hence, SchB-PSA NPs are promising to treat ALI.

Malignant melanoma, a highly dangerous type of skin cancer, is usually resistant to pro-apoptosis agents such as tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) due to low death receptor expression levels. After verifying combination of chemotherapy drug paclitaxel (PTX) and TRAIL could significantly enhance their anti-melanoma effects, we developed a liposomal melanoma target-delivery system with tumor microenvironment responsiveness (TRAIL-[Lip-PTX]C18-TR) to co-deliver TRAIL and PTX. TRAIL is attached to negatively-charged liposome surface while PTX is encapsulated inside, with final surface modification of a stearyl chain (C18) fused pH-sensitive cell-penetrating peptide (TR). Here, C18-TR could specifically binds to melanoma-rich integrin receptors αvβ3 for melanoma targeting, help release TRAIL in low pH microenvironment by reversing the liposomal charge, and facilitate consequent liposome internalization. TRAIL-[Lip-PTX]C18-TR displayed significantly better in vitro half-maximal inhibitory concentration (IC50) than other formulations, and an in vivo tumor inhibition rate of 93.8%. Mechanistic study revealed that this synergistic effect is associated with the upregulation of death receptors DR4/5 by PTX. This co-delivery system significantly improved TRAIL-based therapy against melanoma, and provided a simple platform to co-deliver other drugs/agents for melanoma treatment.

Successful anticancer chemotherapy requires targeting tumors efficiently and further potential to eliminate cancer stem cell (CSC) subpopulations. Since CD44 is present on many types of CSCs, and it binds specially to hyaluronic acid (HA), we tested whether coating solid lipid nanoparticles with hyaluronan (HA-SLNs)would allow targeted delivery of paclitaxel (PTX) to CD44-overexpressing B16F10 melanoma cells. First, we developed a model system based on melanoma stem-like cells for experiments in vitro and in mouse xenografts, and we showed that cells expressing high levels of CD44 (CD44(+)) displayed a strong CSC phenotype while cells expressing low levels of CD44 (CD44(-)) did not. This phenotype included sphere and colony formation, higher proportion of side population cells, expression of CSC-related markers (ALDH, CD133, Oct-4) and tumorigenicity in vivo. Next we showed that administering PTX-loaded HA-SLNs led to efficient intracellular delivery of PTX and induced substantial apoptosis in CD44(+) cells in vitro. In the B16F10-CD44(+) lung metastasis model, PTX-loaded HA-SLNs targeted the tumor-bearing lung tissues well and subsequently exhibited significant antitumor effects with a relative low dose of PTX, which provided significant survival benefit without evidence of adverse events. These findings suggest that the HA-SLNs targeting system shows promise for enhancing cancer therapy.

Our study aimed to develop a self-assembled nanomicelle for oral administration of nimodipine (NIM) with poor water solubility. Using Solutol(®) HS15, the NIM-loaded self-assembled nanomicelles displayed a near-spherical morphology with a narrow size distribution of 12.57 ± 0.21 nm (polydispersity index =0.071 ± 0.011). Compared with Nimotop(®) (NIM tablets), the intestinal absorption of NIM from NIM nanomicelle in rats was improved by 3.13- and 2.25-fold in duodenum and jejunum at 1 hour after oral administration. The cellular transport of NIM nanomicelle in Caco-2 cell monolayers was significantly enhanced compared to that of Nimotop(®). Regarding the transport pathways, clathrin, lipid raft/caveolae, and macropinocytosis mediated the cell uptake of NIM nanomicelles, while P-glycoprotein and endoplasmic reticulum/Golgi complex (ER/Golgi) pathways were involved in exocytosis. Pharmacokinetic studies in our research laboratory have showed that the area under the plasma concentration-time curve (AUC0-∞) of NIM nanomicelles was 3.72-fold that of Nimotop(®) via oral administration in rats. Moreover, the NIM concentration in the brain from NIM nanomicelles was dramatically improved. Therefore, Solutol(®) HS15-based self-assembled nanomicelles represent a promising delivery system to enhance the oral bioavailability of NIM.

A rapid, sensitive and selective liquid chromatography-electrospray ionization-tandem mass spectrometric method was developed and validated for the quantification of scopoletin in rat plasma. After the addition of the internal standard xanthotoxin, plasma samples were pretreated by a simple one-step protein precipitation with acetonitrile-methanol (2:1, v/v). Chromatographic separation was achieved on a Diamonsil ODS chromatography column using gradient elution with the mobile phase consisting of acetonitrile and 0.1% formic acid. The determination was performed by positive ion electrospray ionization in multiple reaction monitoring mode. The calibration curve was linear over the concentration range of 5-1000 ng/mL (r = 0.9996). The intra- and inter-day precision (RSD%) was less than 6.1%, and the accuracy (RE%) was from -3.0%-2.5%. This method was successfully applied to the pharmacokinetic research of scopoletin in rats after intravenous (5 mg/kg) or oral (5, 10 and 20 mg/kg) administration. The result showed that oral bioavailability with a dose of 5 mg/kg was 6.62% ± 1.72%, 10 mg/kg, 5.59% ± 1.16%, and 20 mg/kg, 5.65% ± 0.75%.

Due to its safety and efficacy, aluminum hydroxide is used as an immune adjuvant in human vaccines for over 80 years. Being a Th2 stimulator, the classical gel-like adjuvant, however, fails to generate CD8+ T cell responses, which are important for cancer vaccines. Here, aluminum hydroxide is turned from gel into nano-sized vaccine carriers AlO(OH)-polymer nanoparticles (APNs) to promote their lymphatic migration. After actively uptaken via scavenger receptor-A by antigen-presenting cells (APCs) resident in lymph nodes (LNs), APNs destabilize lysosomes resulting in efficient cytosolic delivery and cross-presentation of antigens. It is demonstrated that administration of APNs loaded with ovalbumin (OVA) and CpG led to the codelivery of both cargos into APCs in LNs, leading to their activation and subsequent adaptive immunity. A prime-boost strategy with low doses of OVA (1.5 µg) and CpG (0.45 µg) induces potent CD8+ T cell responses and dramatically prolongs the survival of B16-OVA tumor-bearing mice. More impressively, when using B16F10 lysates instead of OVA as antigen, substantial antitumor effects on B16F10 tumor model are observed by using APN-CpG. These results suggest the great potential of APNs as vaccine carriers that activate CD8+ T cell responses and the bright prospect of aluminum adjuvant in a nanoparticle formulation.

Vincristine (VCR), which is a widely used antineoplastic drug, was integrated with a submicron-emulsion drug-delivery system to enhance the anticancer effect.

Immunomodulatory effect has been found to be an important therapeutic measure for immune responses against cancer. In this study, we evaluated the inhibition of Scutellaria barbata D. Don (SB), an anti-inflammatory and an antitumor Chinese herb, including flavonoids and scutebarbatines on tumor growth and its immunomodulatory effects in vivo. HPLC and LC/MS/MS methods were conducted for the analysis of flavonoids and scutebarbatines in SB. Lewis-bearing C57BL/6 mice model was established and tumor volume was evaluated by high frequency color ultrasound experiment. ELISA and western blot analysis were performed for the determination of immunomodulatory factors. SB treatment at the dose of 10, 6.67, and 3.33 g crude drug/kg/d significantly inhibited tumor growth of Lewis-bearing C57BL/6 mice with the inhibition rates of 44.41 ± 5.44%, 33.56 ± 4.85%, and 27.57 ± 4.96%, respectively. More importantly, the spleen and thymus indexes were increased remarkably by SB treatment. SB could decrease IL-17, IL-10, FOXP3, TGF-β1, RORγt, and IL-6 levels whereas it could increase remarkably IL-2 and IFN-γ levels. Our results demonstrated that SB could inhibit tumor growth in vivo through regulating immune function in tumor-bearing mice and suggested that the immunomodulatory function of SB had a potential therapeutic effect in lung cancer.

The generation of anti-adenovirus neutralizing antibody (NAb) in humans severely restricts the utilization of recombinant adenovirus serotype 5 (Ad5) vectors in gene therapy for a wide range of clinical trials. To overcome this limitation, we ionically complexed Ad5 with a newly synthesized copolymer, which we called APC, making an adenovirus shielded from NAb.

Currently, most anti-cancer therapies are still haunted by serious and deleterious adverse effects. Here, we report a highly biocompatible tumor cell-targeting delivery systems utilizing exosome-like vesicles (ELVs) that delivers a low-toxicity anti-cancer agent imperialine against non-small cell lung cancer (NSCLC). First, we introduced a novel micelle-aided method to efficiently load imperialine into intact ELVs. Then, integrin α3β1-binding octapeptide cNGQGEQc was modified onto ELV platform for tumor targeting as integrin α3β1 is overexpressed on NSCLC cells. This system not only significantly improved imperialine tumor accumulation and retention, but also had extremely low systemic toxicity both in vitro and in vivo. Our discoveries offer new ways to utilize ELV more efficiently for both drug loading and targeting. The solid pharmacokinetics improvement and extraordinary safety of this system also highlight possibilities of alternative long course cancer therapies using similar strategies.