Selective Intracellular Delivery of Antibodies in Cancer Cells with Nanocarriers Sensing Endo/Lysosomal Enzymatic Activity.

The differential enzymatic activity in the endo/lysosomes of particular cells could trigger targeted endosomal escape functions, enabling selective intracellular protein delivery. However, this strategy may be jeopardized due to protein degradation during endosomal trafficking. Herein, using custom made fluorescent probes to assess the endosomal activity of cathepsin B (CTSB) and protein degradation, we found that certain cancer cells with hyperacidified endosomes grant a spatiotemporal window where CTSB activity surpass protein digestion. This inspired the engineering of antibody-loaded polymeric nanocarriers having CTSB-activatable endosomal escape ability. The nanocarriers selectively escaped from the endo/lysosomes in the cells with high endosomal CTSB activity and delivered active antibodies to intracellular targets. This study provides a viable strategy for cell-specific protein delivery using stimuli-responsive nanocarriers with controlled endosomal escape.

Translational nanomedicine potentiates immunotherapy in sarcoma by normalizing the microenvironment.

Nanocarrier-based chemo-immunotherapy has succeeded in clinical trials and understanding its effect on the tumor microenvironment could facilitate development of strategies to increase efficacy of these regimens further. NC-6300 (epirubicin micelle) demonstrates anti-tumor activity in sarcoma patients, but whether it is combinable with immune checkpoint inhibition is unclear. Here, we tested NC-6300 combined with anti-PD-L1 antibody in mouse models of osteosarcoma and fibrosarcoma. We found that sarcoma responds to NC-6300 in a dose-dependent manner, while anti-PD-L1 efficacy is potentiated even at a dose of NC-6300 less than 10% of the maximum tolerated dose. Furthermore, NC-6300 is more effective than the maximum tolerated dose of doxorubicin in increasing the tumor growth delay induced by anti-PD-L1 antibody. We investigated the mechanism of action of this combination. NC-6300 induces immunogenic cell death and its effect on the efficacy of anti-PD-L1 antibody is dependent on T cells. Also, NC-6300 normalized the tumor microenvironment (i.e., ameliorated pathophysiology towards normal phenotype) as evidenced through increased blood vessel maturity and reduced fibrosis. As a result, the combination with anti-PD-L1 antibody increased the intratumor density and proliferation of T cells. In conclusion, NC-6300 potentiates immune checkpoint inhibition in sarcoma, and normalization of the tumor microenvironment should be investigated when developing nanocarrier-based chemo-immunotherapy regimens.

Conjugation of glucosylated polymer chains to checkpoint blockade antibodies augments their efficacy and specificity for glioblastoma.

Because of the blood-tumour barrier and cross-reactivity with healthy tissues, immune checkpoint blockade therapy against glioblastoma has inadequate efficacy and is associated with a high risk of immune-related adverse events. Here we show that anti-programmed death-ligand 1 antibodies conjugated with multiple poly(ethylene glycol) (PEG) chains functionalized to target glucose transporter 1 (which is overexpressed in brain capillaries) and detaching in the reductive tumour microenvironment augment the potency and safety of checkpoint blockade therapy against glioblastoma. In mice bearing orthotopic glioblastoma tumours, a single dose of glucosylated and multi-PEGylated antibodies reinvigorated antitumour immune responses, induced immunological memory that protected the animals against rechallenge with tumour cells, and suppressed autoimmune responses in the animals' healthy tissues. Drug-delivery formulations leveraging multivalent ligand interactions and the properties of the tumour microenvironment to facilitate the crossing of blood-tumour barriers and increase drug specificity may enhance the efficacy and safety of other antibody-based therapies.

Enzymatically Transformable Polymersome-Based Nanotherapeutics to Eliminate Minimal Relapsable Cancer.

Prevention of metastatic and local-regional recurrence of cancer after surgery remains difficult. Targeting postsurgical premetastatic niche and microresiduals presents an excellent prospective opportunity but is often challenged by poor therapeutic delivery into minimal residual tumors. Here, an enzymatically transformable polymer-based nanotherapeutic approach is presented that exploits matrix metalloproteinase (MMP) overactivation in tumor-associated tissues to guide the codelivery of colchicine (microtubule-disrupting and anti-inflammatory agent) and marimastat (MMP inhibitor). The dePEGylation of polymersomes catalyzed by MMPs not only exposes the guanidine moiety to improve tissue/cell-targeting/retention to increase bioavailability, but also differentially releases marimastat and colchicine to engage their extracellular (MMPs) and intracellular (microtubules) targets of action, respectively. In primary tumors/overt metastases, the vasculature-specific targeting of nanotherapeutics can function synchronously with the enhanced permeability and retention effect to deter malignant progression of metastatic breast cancer. After the surgical removal of large primary tumors, nanotherapeutic agents are localized in the premetastatic niche and at the site of the postsurgical wound, disrupting the premetastatic microenvironment and eliminating microresiduals, which radically reduces metastatic and local-regional recurrence. The findings suggest that nanotherapeutics can safely widen the therapeutic window to resuscitate colchicine and MMP inhibitors for other inflammatory disorders.

Poly-ion complex micelles effectively deliver CoA-conjugated CPT1A inhibitors to modulate lipid metabolism in brain cells.

Carnitine palmitoyltransferase 1A (CPT1A) is a central player in lipid metabolism, catalyzing the first step to fatty acid oxidation (FAO). Inhibiting CPT1A, especially in the brain, can have several pharmacological benefits, such as in treating obesity and brain cancer. C75-CoA is a strong competitive inhibitor of CPT1A. However, due to its negatively charged nature, it has low cellular permeability. Herein, we report the use of poly-ion complex (PIC) micelles to deliver the specific CPT1A inhibitors (±)-, (+)-, and (-)-C75-CoA into U87MG glioma cells and GT1-7 neurons. PIC micelles were formed through charge-neutralization of the cargo with the cationic side chain of PEG-poly{N-[N'-(2-aminoethyl)-2-aminoethyl]aspartamide} (PEG-PAsp(DET)), forming particles with 55 to 65 nm diameter. Upon short-term incubation with cells, the micelle-encapsulated CPT1A inhibitors resulted in up to 5-fold reduction of ATP synthesis compared to the free drug, without an apparent decline in cell viability. Micelle treatment showed a discernible decrease in 14C-palmitate oxidation into CO2 and acid-soluble metabolites, confirming that the substantial lowering of ATP production has resulted from FAO inhibition. Micelle treatment also diminished IC50 by 2 to 4-fold over the free drug-treated U87MG after long-term incubation. To measure the cellular uptake of these CoA-adduct loaded PIC micelles, we synthesized a fluorescent CoA derivative and prepared Fluor-CoA micelles which showed efficient internalization in the cell lines, both in 2D and 3D culture models, especially in neurons where uptake reached up to 3-fold over the free dye. Our results starkly demonstrate that the PIC micelles are a promising delivery platform for anionic inhibitors of CPT1A in glioma cells and neurons, laying the groundwork for future research or clinical applications.

Efficacy of pH-Sensitive Nanomedicines in Tumors with Different c-MYC Expression Depends on the Intratumoral Activation Profile.

Effective inhibition of the protein derived from cellular myelocytomatosis oncogene (c-Myc) is one of the most sought-after goals in cancer therapy. While several c-Myc inhibitors have demonstrated therapeutic potential, inhibiting c-Myc has proven challenging, since c-Myc is essential for normal tissues and tumors may present heterogeneous c-Myc levels demanding contrasting therapeutic strategies. Herein, we developed tumor-targeted nanomedicines capable of treating both tumors with high and low c-Myc levels by adjusting their ability to spatiotemporally control drug action. These nanomedicines loaded homologues of the bromodomain and extraterminal (BET) motif inhibitor JQ1 as epigenetic c-Myc inhibitors through pH-cleavable bonds engineered for fast or slow drug release at intratumoral pH. In tumors with high c-Myc expression, the fast-releasing (FR) nanomedicines suppressed tumor growth more effectively than the slow-releasing (SR) ones, whereas, in the low c-Myc tumors, the efficacy of the nanomedicines was the opposite. By studying the tumor distribution and intratumoral activation of the nanomedicines, we found that, despite SR nanomedicines achieved higher accumulation than the FR counterparts in both c-Myc high and low tumors, the antitumor activity profiles corresponded with the availability of activated drugs inside the tumors. These results indicate the potential of engineered nanomedicines for c-Myc inhibition and spur the idea of precision pH-sensitive nanomedicine based on cancer biomarker levels.

Supramolecularly enabled pH- triggered drug action at tumor microenvironment potentiates nanomedicine efficacy against glioblastoma.

The crucial balance of stability in blood-circulation and tumor-specific delivery has been suggested as one of the challenges for effective bench-to-bedside translation of nanomedicines (NMs). Herein, we developed a supramolecularly enabled tumor-extracellular (Tex) pH-triggered NM that can maintain the micellar structure with the entrapped-drug during systemic circulation and progressively release drug in the tumor by rightly sensing heterogeneous tumor-pH. Desacetylvinblastine hydrazide (DAVBNH), a derivative of potent anticancer drug vinblastine, was conjugated to an aliphatic ketone-functionalized poly(ethylene glycol)-b-poly(amino acid) copolymer and the hydrolytic stability of the derived hydrazone bond was efficiently tailored by exploiting the compartmentalized structure of polymer micelle. We confirmed an effective and safe therapeutic application of Tex pH-sensitive DAVBNH-loaded micelle (Tex-micelle) in orthotopic glioblastoma (GBM) models, extending median survival to 1.4 times in GBM xenograft and 2.6 times in GBM syngeneic model, compared to that of the free DAVBNH. The work presented here offers novel chemical insights into the molecular design of smart NMs correctly sensing Tex-pH via programmed functionalities. The practical engineering strategy based on a clinically relevant NM platform, and the encouraging therapeutic application of Tex-micelle in GBM, one of the most lethal human cancers, thus suggests the potential clinical translation of this system against other types of common cancers, including GBM.

Tumor-Targeted Nanomedicine for Immunotherapy.

Therapeutic manipulation of the immune system against cancer has revolutionized the treatment of several advanced-stage tumors. While many have benefited from these treatments, the proportion of patients responding to immunotherapies is still low. Nanomedicines have promise to revolutionize tumor treatments through spatiotemporal control of drug activity. Such control of drug function could allow enhanced therapeutic actions of immunotherapies and reduced side effects. However, only a handful of formulations have been able to reach human clinical studies so far, and even fewer systems are being used in the clinic. Among translatable formulations, self-assembled nanomedicines have shown unique and versatile features for dealing with the heterogeneity and malignancy of tumors in the clinic. Such nanomedicines can be designed to promote antitumor immune responses through a series of immunopotentiating functions after being directly injected into tumors, or achieving selective tumor accumulation upon intravenous administration. Thus, tumor-targeted nanomedicines can enhance antitumor immunity by several mechanisms, such as inducing immunogenic damage to cancer cells, altering the tumor immune microenvironment by delivering immunomodulators, or eliminating or reprogramming immunosuppressive cells, enhancing the exposure of tumor-associated antigens to antigen presenting cells, stimulating innate immunity mechanisms, and facilitating the infiltration of antitumor immune cells and their interaction with cancer cells. Moreover, nanomedicines can be engineered to sense intratumoral stimuli for activating specific immune responses or installed with ligands for increasing drug levels in tumors, granting subcellular delivery, and triggering immune signals and proliferation of immune cells. Thus, the ability of nanomedicines to exert immunomodulatory functions selectively in tumor and tumor-associated tissues, such as draining lymph nodes, increases the efficiency of the treatments, while avoiding systemic immunosuppressive toxicities and the exacerbation of adverse immune responses. Moreover, the compartmentalized structure of self-assembled nanomedicines offers the possibility to coload a variety of drugs for controlled pharmacokinetics, enhanced tumor delivery, and synergistic therapeutic output. Also, by integrating imaging functionalities into nanomedicines, it is possible to develop theranostic platforms reporting the immune settings of tumors as well as the effects of nanomedicines on the tumor immune microenvironment. Herein, we critically reviewed significant strategies for developing nanomedicines capable of potentiating antitumor immune responses by surmounting biological barriers and modulating antitumor immune signals. Moreover, the potential of these nanomedicines for developing innovative anticancer treatments by targeting particular cells is discussed. Finally, we present our perspectives on the awaiting challenges and future directions of nanomedicines in the age of immunotherapy.

Translational Nanomedicine Boosts Anti-PD1 Therapy to Eradicate Orthotopic PTEN-Negative Glioblastoma.

Glioblastoma (GBM) is resistant to immune checkpoint inhibition due to its low mutation rate, phosphatase and tensin homologue (PTEN)-deficient immunosuppressive microenvironment, and high fraction of cancer stem-like cells (CSCs). Nanomedicines fostering immunoactivating intratumoral signals could reverse GBM resistance to immune checkpoint inhibitors (ICIs) for promoting curative responses. Here, we applied pH-sensitive epirubicin-loaded micellar nanomedicines, which are under clinical evaluation, to synergize the efficacy of anti-PD1antibodies (aPD1) against PTEN-positive and PTEN-negative orthotopic GBM, the latter with a large subpopulation of CSCs. The combination of epirubicin-loaded micelles (Epi/m) with aPD1 overcame GBM resistance to ICIs by transforming cold GBM into hot tumors with high infiltration of antitumor immune cells through the induction of immunogenic cell death (ICD), elimination of immunosuppressive myeloid-derived suppressor cells (MSDCs), and reduction of PD-L1 expression on tumor cells. Thus, Epi/m plus aPD1 eradicated both PTEN-positive and PTEN-negative orthotopic GBM and provided long-term immune memory effects. Our results indicate the high translatable potential of Epi/m plus aPD1 for the treatment of GBM.

Transient stealth coating of liver sinusoidal wall by anchoring two-armed PEG for retargeting nanomedicines.

A major critical issue in systemically administered nanomedicines is nonspecific clearance by the liver sinusoidal endothelium, causing a substantial decrease in the delivery efficiency of nanomedicines into the target tissues. Here, we addressed this issue by in situ stealth coating of liver sinusoids using linear or two-armed poly(ethylene glycol) (PEG)-conjugated oligo(l-lysine) (OligoLys). PEG-OligoLys selectively attached to liver sinusoids for PEG coating, leaving the endothelium of other tissues uncoated and, thus, accessible to the nanomedicines. Furthermore, OligoLys having a two-armed PEG configuration was ultimately cleared from sinusoidal walls to the bile, while OligoLys with linear PEG persisted in the sinusoidal walls, possibly causing prolonged disturbance of liver physiological functions. Such transient and selective stealth coating of liver sinusoids by two-arm-PEG-OligoLys was effective in preventing the sinusoidal clearance of nonviral and viral gene vectors, representatives of synthetic and nature-derived nanomedicines, respectively, thereby boosting their gene transfection efficiency in the target tissues.

In Vitro Blood-Brain Barrier Permeability and Cytotoxicity of an Atorvastatin-Loaded Nanoformulation Against Glioblastoma in 2D and 3D Models.

Inhibitors of 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase of the family of statins have been suggested as therapeutic options in various tumors. Atorvastatin is a statin with the potential to cross the blood-brain barrier; however, the concentrations necessary for a cytotoxic effect against cancer cells exceed the concentrations achievable via oral administration, which made the development of a novel atorvastatin formulation necessary. We characterized the drug loading and basic physicochemical characteristics of micellar atorvastatin formulations and tested their cytotoxicity against a panel of different glioblastoma cell lines. In addition, activity against tumor spheroids formed from mouse glioma and mouse cancer stem cells, respectively, was evaluated. Our results show good activity of atorvastatin against all tested cell lines. Interestingly, in the three-dimensional (3D) models, growth inhibition was more pronounced for the micellar formulation compared to free atorvastatin. Finally, atorvastatin penetration across a blood-brain barrier model obtained from human induced-pluripotent stem cells was evaluated. Our results suggest that the presented micelles may enable much higher serum concentrations than possible by oral administration; however, if transport across the blood-brain barrier is sufficient to reach the therapeutic atorvastatin concentration for the treatment of glioblastoma via intravenous administration remains unclear.

Nanomedicines blocking adaptive signals in cancer cells overcome tumor TKI resistance.

Tumor resistance to tyrosine kinase inhibitors (TKIs) is an inexorable clinical event. The manipulation of adaptive changes in cancer cells while inhibiting the signaling pathways could be an effective strategy for overcoming TKI resistance toward reducing tumor relapse and prolonging survival. Here, we tested this approach by using polymeric nanomedicines delivering the pan-kinase inhibitor staurosporine (STS) to treat renal cell carcinoma (RCC) resistant to the multi-targeted TKI sunitinib. STS blocked the activity of TKI-resistant protein kinases and strongly inhibited adaptive dynamics in RCC cells promoted by MDR1 and GLUT1 to overcome sunitinib resistance. Co-delivery of STS and epirubicin directed to eliminate fast-proliferating cancer cells through the same nanomedicine platform enabled safe and potent in vivo efficacy in mouse models of RCC, overcoming sunitinib resistance and suppressing the development of metastasis. These results indicate our approach as a promising strategy for effectively managing TKI resistance.

Single-Stranded DNA-Packaged Polyplex Micelle as Adeno-Associated-Virus-Inspired Compact Vector to Systemically Target Stroma-Rich Pancreatic Cancer.

Despite the rigidity of double-stranded DNA (dsDNA), its packaging is used to construct nonviral gene carriers due to its availability and the importance of its double-helix to elicit transcription. However, there is an increasing demand for more compact-sized carriers to facilitate tissue penetration, which may be easily fulfilled by using the more flexible single-stranded DNA (ssDNA) as an alternative template. Inspired by the adeno-associated virus (AAV) as a prime example of a transcriptionally active ssDNA system, we considered a methodology that can capture unpaired ssDNA within the polyplex micelle system (PM), an assembly of DNA and poly(ethylene glycol)-b-poly(l-lysine) (PEG-PLys). A micellar assembly retaining unpaired ssDNA was prepared by unpairing linearized pDNA with heat and performing polyion complexation on site with PEG-PLys. The PM thus formed had a compact and spherical shape, which was distinguishable from the rod-shaped PM formed from dsDNA, and still retained its ability to activate gene expression. Furthermore, we demonstrated that its capacity to encapsulate DNA was much higher than AAV, thereby potentially allowing the delivery of a larger variety of protein-encoding DNA. These features permit the ssDNA-loaded PM to easily penetrate the size-restricting stromal barrier after systemic application. Further, they can elicit gene expression in tumor cell nests of an intractable pancreatic cancer mouse model to achieve antitumor effects through suicide gene therapy. Thus, single-stranded DNA-packaged PM is appealing as a potential gene vector to tackle intractable diseases, particularly those with target delivery issues due to size-restriction barriers.

In vivo rendezvous of small nucleic acid drugs with charge-matched block catiomers to target cancers.

Stabilisation of fragile oligonucleotides, typically small interfering RNA (siRNA), is one of the most critical issues for oligonucleotide therapeutics. Many previous studies encapsulated oligonucleotides into ~100-nm nanoparticles. However, such nanoparticles inevitably accumulate in liver and spleen. Further, some intractable cancers, e.g., tumours in pancreas and brain, have inherent barrier characteristics preventing the penetration of such nanoparticles into tumour microenvironments. Herein, we report an alternative approach to cancer-targeted oligonucleotide delivery using a Y-shaped block catiomer (YBC) with precisely regulated chain length. Notably, the number of positive charges in YBC is adjusted to match that of negative charges in each oligonucleotide strand (i.e., 20). The YBC rendezvouses with a single oligonucleotide in the bloodstream to generate a dynamic ion-pair, termed unit polyion complex (uPIC). Owing to both significant longevity in the bloodstream and appreciably small size (~18 nm), the uPIC efficiently delivers oligonucleotides into pancreatic tumour and brain tumour models, exerting significant antitumour activity.

Therapeutic Polymersome Nanoreactors with Tumor-Specific Activable Cascade Reactions for Cooperative Cancer Therapy.

Therapeutic nanoreactors are of increasing interest in precise cancer therapy, which have been explored to in situ produce therapeutic compounds from inert prodrugs or intrinsic molecules at the target sites. However, engineering a nanoreactor with tumor activable cascade reactions for efficient cooperative cancer therapy remains a great challenge. Herein, we demonstrate a polymersome nanoreactor with tumor acidity-responsive membrane permeability to activate cascade reactions for orchestrated cooperative cancer treatment. The nanoreactors are constructed from responsive polyprodrug polymersomes incorporating ultrasmall iron oxide nanoparticles and glucose oxidase in the membranes and inner aqueous cavities, respectively. The cascade reactions including glucose consumption to generate H2O2, accelerated iron ion release, Fenton reaction between H2O2 and iron ion to produce hydroxyl radicals (•OH), and •OH-triggered rapid release of parent drugs can be specifically activated by the tumor acidity-responsive membrane permeability. During this process, the orchestrated cooperative cancer therapy including starving therapy, chemodynamic therapy, and chemotherapy is realized for high-efficiency tumor suppression by the in situ consumed and produced compounds. The nanoreactor design with tumor-activable cascade reactions represents an insightful paradigm for precise cooperative cancer therapy.

Glucose-linked sub-50-nm unimer polyion complex-assembled gold nanoparticles for targeted siRNA delivery to glucose transporter 1-overexpressing breast cancer stem-like cells.

Cancer stem-like cells (CSCs) treatment is a plausible strategy for enhanced cancer therapy. Here we report a glucose-installed sub-50-nm nanocarrier for the targeted delivery of small interfering RNA (siRNA) to CSCs through selective recognition of the glucose ligand to the glucose transporter 1 (GLUT1) overexpressed on the CSC surface. The siRNA nanocarrier was constructed via a two-step assembling process. First, a glucose-installed poly(ethylene glycol)-block-poly(l-lysine) modified with lipoic acid (LA) at the ω-end (Glu-PEG-PLL-LA) was associated with a single siRNA to form a unimer polyion complex (uPIC). Second, a 20 nm gold nanoparticle (AuNP) was decorated with ~65 uPICs through AuS bonding. The glucose-installed targeted nanoparticles (Glu-NPs) exhibited higher cellular uptake of siRNA payloads in a spheroid breast cancer (MBA-MB-231) cell culture compared with glucose-unconjugated control nanoparticles (MeO-NPs). Notably, the Glu-NPs became more efficiently internalized into the CSC fraction, which was defined by aldehyde dehydrogenase (ALDH) activity assay, than the other fractions, probably due to the higher GLUT1 expression level on the CSCs. The Glu-NPs elicited significantly enhanced gene silencing in a CSC-rich orthotopic MDA-MB-231 tumor tissue following systemic administration to tumor-bearing mice. Ultimately, the repeated administrations of polo-like kinase 1 (PLK1) siRNA-loaded Glu-NPs significantly suppressed the growth of orthotopic MDA-MB-231 tumors. These results demonstrate that Glu-NP is a promising nanocarrier design for CSC-targeted cancer treatment.

Effective treatment of drug resistant recurrent breast tumors harboring cancer stem-like cells by staurosporine/epirubicin co-loaded polymeric micelles.

Breast cancer recurrence and resistance are associated with cancer stem-like cell (CSC) sub-populations. As conventional therapies fail to treat CSCs, institution of novel therapeutic strategies capable of eradicating both cancer cells and CSCs is central for achieving effective treatments with long-term survival. Here, we studied the ability of polymeric micelles cooperatively loading the cytotoxic drug epirubicin (Epi) and the CSC inhibitor staurosporine (STS) to treat breast tumors, particularly when tumors relapsed after chemotherapy. The STS/Epi-loaded micelles (STS/Epi/m) demonstrated potent therapeutic efficacy against both naïve orthotopic 4T1-luc breast tumors and their recurrent Epi-resistant counterparts, significantly prolonging survival. This efficacy enhancement of STS/Epi/m was correlated with the ability of the micelles to suppress the CSC-associated sub-populations of breast cancer, i.e. the aldehyde dehydrogenase-positive (ALDH+) population and the CD44+/CD24- fraction, in Epi-resistant cells and tumors. These results demonstrated STS/Epi/m as a promising strategy for effective management of breast cancer.

cRGD peptide installation on cisplatin-loaded nanomedicines enhances efficacy against locally advanced head and neck squamous cell carcinoma bearing cancer stem-like cells.

Recalcitrant head and neck squamous cell carcinoma (HNSCC) usually relapses after therapy due to the enrichment of drug resistant cancer stem-like cells (CSCs). Nanomedicines have shown potential for eradicating both cancer cells and CSCs by effective intratumoral navigation for reaching particular cell populations and controlling drug delivery. The installation of ligands on nanomedicines is an attractive approach for improving the delivery to CSCs within tumors, though the development of CSC-selective ligand-receptor systems has been challenging. Herein, we found that the CSC subpopulation in HNSCC cells overexpresses αvß5 integrins, which is preferentially expressed in tumor neovasculature and cancer cells, and can be effectively targeted by using cyclic Arg-Gly-Asp (cRGD) peptide. Thus, in this study, we propose installing cRGD peptide on micellar nanomedicines incorporating cisplatin for improving their activity against CSCs and enhancing survival. Both cisplatin-loaded micelles (CDDP/m) and cRGD-installed CDDP/m (cRGD-CDDP/m) were effective against HNSCC SAS-L1-Luc cells in vitro, though cRGD-installed CDDP/m was more potent than CDDP/m against the CSC fraction. In vivo, the cRGD-CDDP/m also showed significant antitumor activity against HNSCC orthotopic tumors, i.e. SAS-L1 and HSC-2. Moreover, cRGD-CDDP/m rapidly accumulated into the lymph node metastasis of SAS-L1 tumors, effectively inhibiting their growth, and prolonging mice survival. These findings indicate cRGD-installed nanomedicines as an advantageous strategy for targeting CSCs in HNSCC, and particularly, cRGD-CDDP/m as a significant therapeutic strategy against regionally advanced HNSCC.

Nanomedicines Eradicating Cancer Stem-like Cells in Vivo by pH-Triggered Intracellular Cooperative Action of Loaded Drugs.

Nanomedicines capable of control over drug functions have potential for developing resilient therapies, even against tumors harboring recalcitrant cancer stem cells (CSCs). By coordinating drug interactions within the confined inner compartment of core-shell nanomedicines, we conceived multicomponent nanomedicines directed to achieve synchronized and synergistic drug cooperation within tumor cells as a strategy for enhancing efficacy, overcoming drug resistance, and eradicating CSCs. The approach was validated by using polymeric micellar nanomedicines co-incorporating the pan-kinase inhibitor staurosporine (STS), which was identified as the most potent CSC inhibitor from a panel of signaling-pathway inhibitors, and the cytotoxic agent epirubicin (Epi), through rationally contriving the affinity between the drugs. The micelles released both drugs simultaneously, triggered by acidic endosomal pH, attaining concurrent intracellular delivery, with STS working as a companion for Epi, down-regulating efflux transporters and resistance mechanisms induced by Epi. These features prompted the nanomedicines to eradicate orthotopic xenografts of Epi-resistant mesothelioma bearing a CSC subpopulation.

Systemic delivery of messenger RNA for the treatment of pancreatic cancer using polyplex nanomicelles with a cholesterol moiety.

Systemic delivery of messenger RNA (mRNA) is technically challenging because mRNA is highly susceptible to enzymatic degradation in the blood circulation. In this study, we used a nanomicelle-based platform, prepared from mRNA and poly(ethylene glycol) (PEG)-polycation block copolymers. A cholesterol (Chol) moiety was attached to the ω-terminus of the block copolymer to increase the stability of the nanomicelle by hydrophobic interaction. After in vitro screening, polyaspartamide with four aminoethylene repeats in its side chain (PAsp(TEP)) was selected as the cationic segment of the block copolymer, because it contributes to enhance nuclease resistance and high protein expression from the mRNA. After intravenous injection, PEG-PAsp(TEP)-Chol nanomicelles showed significantly enhanced blood retention of mRNA in comparison to nanomicelles without Chol. We used the nanomicelles for treating intractable pancreatic cancer in a subcutaneous inoculation mouse model through the delivery of mRNA encoding an anti-angiogenic protein (sFlt-1). PEG-PAsp(TEP)-Chol nanomicelles generated efficient protein expression from the delivered mRNA in tumor tissue, resulting in remarkable inhibition of the tumor growth, whereas nanomicelles without Chol failed to show a detectable therapeutic effect. In conclusion, the stabilized nanomicelle system led to the successful systemic delivery of mRNA in therapeutic application, holding great promise for the treatment of various diseases.