Engineering Lipusu with lysophosphatidylcholine for improved tumor cellular uptake and anticancer efficacy.

Liposomes have been developed as drug delivery carriers to enhance the antitumor efficiency of therapeutic agents. Lipusu® (Lip), a paclitaxel (PTX) liposome, has been widely used in the treatment of breast cancer. Compared with PTX, Lip could change the biodistribution and reduce the systemic toxicity. However, there was no positive effect on the entry of PTX into tumor cells, and thus the therapeutic effect was not significantly improved. Therefore, it is meaningful to engineer Lip for improving tumor cellular uptake efficiency. Here, lysophosphatidylcholine (LPC)-engineered Lip (LPC-Lip) was constructed via inserting single chain lipid tails into liposomal lipid bilayers, which was realized by simple incubation. Compared with Lip, the better cellular uptake of liposomes modified with LPC resulted in enhanced cytotoxic activity of LPC-Lip in 4T1 cells. Furthermore, stronger tumor growth inhibition was observed in LPC-Lip treated 4T1 tumor-bearing mice without significant side effects. In conclusion, by modulating the lipid composition of Lip, the antitumor efficacy can be improved, and LPC engineered Lip may serve as a promising formulation of PTX for future cancer therapy.

Development of transferrin functionalized poly(ethylene glycol)/poly(lactic acid) amphiphilic block copolymeric micelles as a potential delivery system targeting brain glioma.

The aim of present study is to conceive a biodegradable poly(ethylene glycol)-polylactide (PEG-PLA) copolymer nanoparticle which can be surface biofunctionalized with ligands via biotin-avidin interactions and used as a potential drug delivery carrier targeting to brain glioma in vivo. For this aim, a new method was employed to synthesize biotinylated PEG-PLA copolymers, i.e., esterification of PEG with biotinyl chloride followed by copolymerization of hetero-biotinylated PEG with lactide. PEG-PLA nanoparticles bearing biotin groups on surface were prepared by nanoprecipitation technique and the functional protein transferrin (Tf) were coupled to the nanoparticles by taking advantage of the strong biotin-avidin complex formation. The flow cytometer measurement demonstrated the targeting ability of the nanoparticles to tumor cells in vitro, and the fluorescence microscopy observation of brain sections from C6 glioma tumor-bearing rat model gave the intuitive proof that Tf functionalized PEG-PLA nanoparticles could penetrate into tumor in vivo.

Growth inhibition against intracranial C6 glioma cells by stereotactic delivery of BCNU by controlled release from poly(D,L-lactic acid) nanoparticles.

Transferrin (Tf), an iron-transporting serum glycoprotein, which binds to receptors expressed at the surface of most proliferating cells with particularly high expression on erythroblasts and cancer cells, was chosen as the ligand to develop BCNU-loaded biodegradable poly(D,L-lactic acid) nanoparticles (NPs) containing a ligand, which specifically binds to glioma cells, and their anti-tumor ability was evaluated using a C6 glioma model. In vitro drug release behavior demonstrated that BCNU-loaded PLA NPs show certain sustained release characteristics. NPs with low molecular weight PLA showed a higher burst effect and a significantly faster drug release from PLA samples. The biodistribution of Tf-coated nanoparticles investigated by 99Tc-labeled SPECT showed that the surface-containing transferrin PLA nanoparticles were concentrated in the brain and no radioactive foci could be found outside the brain. Inhibition of tumor growth in the C6 tumor-bearing animal model showed that BCNU-loaded PLA NPs had stronger cytotoxicity and prolonged the average survival time of rats. Especially when treated at an early stage with a higher dosage of NPs, the average survival time of rats was prolonged 88.37%. Furthermore, one rat maintained normal behavior continuously for an observation period of up to 60 days.

Dual-Locking Nanoparticles Disrupt the PD-1/PD-L1 Pathway for Efficient Cancer Immunotherapy.

The clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated (Cas) enzyme, Cas13a, holds great promise in cancer treatment due to its potential for selective destruction of tumor cells via collateral effects after target recognition. However, these collateral effects do not specifically target tumor cells and may cause safety issues when administered systemically. Herein, a dual-locking nanoparticle (DLNP) that can restrict CRISPR/Cas13a activation to tumor tissues is described. DLNP has a core-shell structure, in which the CRISPR/Cas13a system (plasmid DNA, pDNA) is encapsulated inside the core with a dual-responsive polymer layer. This polymer layer endows the DLNP with enhanced stability during blood circulation or in normal tissues and facilitates cellular internalization of the CRISPR/Cas13a system and activation of gene editing upon entry into tumor tissue. After carefully screening and optimizing the CRISPR RNA (crRNA) sequence that targets programmed death-ligand 1 (PD-L1), DLNP demonstrates the effective activation of T-cell-mediated antitumor immunity and the reshaping of immunosuppressive tumor microenvironment (TME) in B16F10-bearing mice, resulting in significantly enhanced antitumor effect and improved survival rate. Further development by replacing the specific crRNA of target genes can potentially make DLNP a universal platform for the rapid development of safe and efficient cancer immunotherapies.

In Situ Modification of the Tumor Cell Surface with Immunomodulating Nanoparticles for Effective Suppression of Tumor Growth in Mice.

Current cancer immunotherapies including chimeric antigen receptor (CAR)-based therapies and checkpoint immune inhibitors have demonstrated significant clinical success, but always suffer from immunotoxicity and autoimmune disease. Recently, nanomaterial-based immunotherapies are developed to precisely control in vivo immune activation in tumor tissues for reducing immune-related adverse events. However, little consideration has been put on the spatial modulation of interactions between immune cells and cancer cells to optimize the efficacy of cancer immunotherapies. Herein, a rational design of immunomodulating nanoparticles is demonstrated that can in situ modify the tumor cell surface with natural killer cell (NK cell)-activating signals to achieve in situ activation of tumor-infiltrating NK cells, as well as direction of their antitumor immunity toward tumor cells. Using these immunomodulating nanoparticles, the remarkable inhibition of tumor growth is observed in mice without noticeable side effects. This study provides an accurate immunomodulation strategy that achieves safe and effective antitumor immunity through in situ NK cell activation in tumors. Further development by constructing interactions with various immune cells can potentially make this nanotechnology become a general platform for the design of advanced immunotherapies for cancer treatments.

A Bioinspired Platform for Effective Delivery of Protein Therapeutics to the Central Nervous System.

Central nervous system (CNS) diseases are the leading cause of morbidity and mortality; their treatment, however, remains constrained by the blood-brain barrier (BBB) that impedes the access of most therapeutics to the brain. A CNS delivery platform for protein therapeutics, which is achieved by encapsulating the proteins within nanocapsules that contain choline and acetylcholine analogues, is reported herein. Mediated by nicotinic acetylcholine receptors and choline transporters, such nanocapsules can effectively penetrate the BBB and deliver the therapeutics to the CNS, as demonstrated in mice and non-human primates. This universal platform, in general, enables the delivery of any protein therapeutics of interest to the brain, opening a new avenue for the treatment of CNS diseases.

Star-branched amphiphilic PLA-b-PDMAEMA copolymers for co-delivery of miR-21 inhibitor and doxorubicin to treat glioma.

The combined treatment of chemotherapeutant and microRNA (miR) has been proven to be a viable strategy for enhancing chemosensitivity due to its synergistic effect for tumor therapy. However, the co-delivery of drugs and genes remains a major challenge as they lack efficient co-delivery carriers. In this study, three amphiphilic star-branched copolymers comprising polylactic acid (PLA) and polydimethylaminoethyl methacrylate (PDMAEMA) with AB3, (AB3)2,and (AB3)3 molecular architectures were synthesized respectively by a combination of ring-opening polymerization, atom transfer radical polymerization, and click chemistry via an "arm-first" approach. The star copolymers possessed a low critical micelle concentration (CMC) and formed nano-sized micelles with positive surface charges in water as well as exhibiting a much lower cytotoxicity than PEI 25 kDa. Nevertheless, their gene transfection efficiency and tumor inhibition ability showed a remarkable dependence on their molecular architecture. The (AB3)3 architecture micelle copolymer exhibited the highest transfection efficiency, about 2.5 times higher than PEI. In addition, after co-delivering DOX and miR-21 inhibitor (miR-21i) into LN229 glioma cells, the micelles could mediate escaping miR-21i from lysosome degradation and the release of DOX to the nucleus, which significantly decreased the miR-21 expression. Moreover, co-delivery of DOX and miR-21i surprisingly exhibited an anti-proliferative efficiency compared with DOX or the miR-21i treatment alone. These results demonstrated that amphiphilic star-branched copolymers are highly promising for their combinatorial delivery of genes and hydrophobic therapeutants.

Smart multifunctional core-shell nanospheres with drug and gene co-loaded for enhancing the therapeutic effect in a rat intracranial tumor model.

Glioblastoma with high mortality has been one of the most serious cancers threatening human health. Because of the present treatment limitations, there is an urgent need to construct a multifunctional vesicle for enhancing the treatment of in situ malignant glioblastoma. In our study, drug and gene co-loaded magnetic PLGA/multifunctional polymeric liposome (magnetic PLGA/MPLs) core-shell nanospheres were constructed. They were mainly self-assembled from two parts: hydrophobic PLGA cores that can load drugs and magnetic nanocrystals; and polymeric lipid shells anchored with functional molecules such as PEG chains, TAT peptides and RGD peptides that can help the vectors to condense the gene, prolong the circulation time, cross the blood brain barrier and target delivery to the cancer tissue. The results showed that the magnetic PLGA/MPLs nanosphere has a nanosized core-shell structure, can achieve sustained drug release and has good DNA binding abilities. Importantly, compared with the control group and other groups with single functionality, it can co-deliver the drug and gene into the same cell in vitro and show the strongest inhibiting effect on the growth of the in situ malignant glioblastoma in vivo. All of these results indicated that the different functional components of magnetic PLGA/MPLs, can form an organic whole and none of them can be dispensed with. The magnetic PLGA/MPLs nanosphere may be another option for treatment of glioblastoma.

Evaluation of folate-PAMAM for the delivery of antisense oligonucleotides to rat C6 glioma cells in vitro and in vivo.

In the current study, we evaluated the efficiency of folate-polyamidoamine dendrimers conjugates (FA-PAMAM) for the in situ delivery of therapeutic antisense oligonucleotides (ASODN) that could inhibit the growth of C6 glioma cells. Folic acid was coupled to the surface amino groups of G5-PAMAM dendrimer (G5D) through a 1-[3-(dimethylamino)propyl]-3-ethylcarbodiimide bond, and ASODNs corresponding to rat epidermal growth factor receptor (EGFR) were then complexed with FA-PAMAM. At an ASODN to PAMAM ratio of 16:1, agarose electrophoresis indicated that antisense oligonucleotides were completely complexed with PAMAM or FA-PAMAM. The ASODN transfection rates mediated by FA-PAMAM and PAMAM were superior to oligofectamine, resulting in greater suppression of EGFR expression and glioma cell growth. Stereotactic injection of EGFR ASODN:FA-PAMAM complexes into established rat C6 intracranial gliomas resulted in greater suppression of tumor growth and longer survival time of tumor-bearing rats compared with PAMAM and oligofectamine-mediated EGFR-ASODN therapy. The current study demonstrates the suitability of folate-PAMAM dendrimer conjugates for efficient EGFR ASODN delivery into glioma cells, wherein they release the ASODN from the FA-PAMAM to knock down EGFR expression in C6 glioma cells, both in vitro and in vivo. FA-PAMAM may thus represent a novel delivery system for short oligonucleotides in glioma-targeted therapy.

Characterization of endocytosis of transferrin-coated PLGA nanoparticles by the blood-brain barrier.

Many studies showed that transferrin increases brain delivery of nanoparticles (NPs) in vivo, however the mechanisms implied in their brain uptake are not yet clearly elucidated. In this study we evaluated the endocytosis of PLGA NPs coated with transferrin on an in vitro model of the blood-brain barrier (BBB) made of a co-culture of brain endothelial cells and astrocytes. PLGA NPs were prepared using DiI as a fluorescent marker and coated with Tween 20, BSA and transferrin (Tf). Blank and BSA-NPs served as controls. The cellular toxicity on BBB of the different samples was evaluated following tight junction aperture and due to high toxicity NPs prepared with Tween 20 were discarded. The size of the NPs prepared by the solvent diffusion method, varied from 63 to 90 nm depending on DiI incorporation and surface coating. Proteins adsorption on the surface of the NPs was found to be stable for at least 12 days at 37 degrees C. Contrary to Blank or BSA-NPs, Tf-NPs were found to be highly adsorbed by the cells and endocytosed using an energy-dependent process. Studies in presence of inhibitors suggest that Tf-NPs interact with the cells in a specific manner and enter the cells via the caveolae pathway.