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Novel approaches are required to address the urgent need to develop lipid-based carriers of paclitaxel (PTX) and other hydrophobic drugs for cancer chemotherapy. Carriers based on cationic liposomes (CLs) with fluid (i.e., chain-melted) membranes (e.g., EndoTAG-1®) have shown promise in preclinical and late-stage clinical studies. Recent work found that the addition of a cone-shaped poly(ethylene glycol)-lipid (PEG-lipid) to PTX-loaded CLs (CLsPTX) promotes a transition to sterically stabilized, higher-curvature (smaller) nanoparticles consisting of a mixture of PEGylated CLsPTX and PTX-containing fluid lipid nanodiscs (nanodiscsPTX). These CLsPTX and nanodiscsPTX show significantly improved uptake and cytotoxicity in cultured human cancer cells at PEG coverage in the brush regime (10 mol% PEG-lipid). Here, we studied the PTX loading, in vivo circulation half-life, and biodistribution of systemically administered CLsPTX and nanodiscsPTX and assessed their ability to induce apoptosis in triple-negative breast cancer-bearing immunocompetent mice. We focused on fluid rather than solid lipid nanodiscs because of the significantly higher solubility of PTX in fluid membranes. At 5 and 10 mol% of a PEG-lipid (PEG5K-lipid, molecular weight of PEG 5000 g/mol), the mixture of PEGylated CLsPTX and nanodiscsPTX was able to incorporate up to 2.5 mol% PTX without crystallization for at least 20 h. Remarkably, compared to preparations containing 2 and 5 mol% PEG5K-lipid (with the PEG chains in the mushroom regime), the particles at 10 mol% (with PEG chains in the brush regime) showed significantly higher blood half-life, tumor penetration and proapoptotic activity. Our study suggests that increasing the PEG coverage of CL-based drug nanoformulations can improve their pharmacokinetics and therapeutic efficacy., This research study was supported by the National Institutes of Health under award R01GM130769 (CRS, KKE, WF; mechanistic studies on developing lipid nanoparticles for drug delivery), the European Regional Development Fund (TT, Project No. 2014-2020.4.01.15-0012), the Estonian Research Council (TT, grants PRG230 and EAG79; PS, grant PSG38; LSG, grant MOBJD11), EuronanomedII projects ECM-CART and iNanoGun (TT), H2020 MSCA-RISE project Oxigenated (TT), and the Spanish Ministry of Science and Innovation grants RYC2020-028754-I and PID2021-122364OA-I00 (PS). Partial support was provided by the US National Science Foundation (NSF) under Award DMR-1807327 (CRS; kinetic phase behavior of cationic vesicles with incorporated hydrophobic molecules).

Myocardial infarction (MI) is the leading cause of death worldwide. However, current therapies are unable to restore the function of the injured myocardium. Advanced approaches, such as stimulation of cardiomyocyte (CM) proliferation are promising, but suffer from poor pharmacokinetics and possible systemic adverse effects. Nanomedicines can be a solution to the above-mentioned drawbacks. However, targeting the cardiac tissue still represents a challenge. Herein, a MI-selective precision nanosystem is developed, that relies on the heart targeting properties of atrial natriuretic peptide (ANP) and lin-TT1 peptide-mediated hitchhiking on M2-like macrophages. The system based on pH-responsive putrescine-modified acetalated dextran (Putre-AcDEX) nanoparticles, shows biocompatibility with cultured cardiac cells, and ANP receptor-dependent interaction with CMs. Moreover, treatment with nanoparticles (NPs) loaded with two pleiotropic cellular self-renewal promoting compounds, CHIR99021 and SB203580, induces a 4-fold increase in bromodeoxyuridine (BrdU) incorporation in primary cardiomyocytes compared to control. In vivo studies confirm that M2-like macrophages targeting by lin-TT1 peptide enhances the heart targeting of ANP. In addition, NP administration does not alter the immunological profile of blood and spleen, showing the short-term safety of the developed system in vivo. Overall, the study results in the development of a peptide-guided precision nanosystem for delivery of therapeutic compounds to the infarcted heart.

Although many studies have explored the depletion of tumor-associated macrophages (TAM) as a therapeutic strategy for solid tumors, currently available compounds suffer from poor efficacy and dose-limiting side effects. Here, we developed a novel TAM-depleting agent (“OximUNO”) that specifically targets CD206+ TAMs and demonstrated efficacy in a triple-negative breast cancer (TNBC) mouse model. OximUNO comprises a star-shaped polyglutamate (St-PGA) decorated with the CD206-targeting peptide mUNO that carries the chemotherapeutic drug doxorubicin (DOX). In the TNBC model, a fluorescently labeled mUNO-decorated St-PGA homed to CD206+ TAMs within primary lesions and metastases. OximUNO exhibited no acute liver or kidney toxicity in vivo. Treatment with OximUNO reduced the progression of primary tumor lesions and pulmonary metastases, significantly diminished the number of CD206+ TAMs and increased the CD8/FOXP3 expression ratio (indicating immunomodulation). Our findings suggest the potential benefit of OximUNO as a TAM-depleting agent for TNBC treatment. Importantly, our studies also represent a novel design of a peptide-targeted St-PGA as a targeted therapeutic nanoconjugate.Significance:A peptide-targeted nanoformulation of DOX exclusively eliminates mannose receptor+ TAMs in breast cancer models, generating response without off-target effects (a drawback of many TAM-depleting agents under clinical study).

One .docx word file containing the following information:Scheme S1. Synthetic approach for St-PGA-OG-mUNO.Scheme S2. Synthetic approach for OximUNO.Video S1. Fifty ns MD trajectory of St-PGA in solution.Table S1. St-PGA-OG-mUNO and St-PGA-OG dye loading, mUNO loading, size and charge.Table S2. OximUNO and St-PGA-DOX drug loading, mUNO loading, size and charge.Table S3. Hepatic and renal toxicity analysed following one dose of OximUNO.Fig. S1. Representative characterisation of St-PGA-OG and St-PGA-OG-mUNO.Fig. S2. The angle used to characterise the rotation of mUNO around PGA.Fig. S3. St-PGA-OG-mUNO shows low hepatic accumulation in the orthotopic TNBC.Fig. S4. Immunostaining showing CD206 expression in the liver compared to the saturated signal of CD206 in the orthotopic 4T1 tumour.Fig. S5. Immunostaining of endogenous IgG indicates leaky tumour vasculature in the orthotopic 4T1 tumours.Fig. S6. Distribution of St-PGA-OG-mUNO in the lung, spleen, sentinel lymph node (SLN) and kidney.Fig. S7. St-PGA-OG-mUNO shows low hepatic accumulation in the experimental metastasis of TNBC.Fig. S8. St-PGA-OG-mUNO shows higher receptor colocalisation than FAM-mUNO.Fig. S9. St-PGA-OG-mUNO shows high homing to M2 TAMs in the orthotopic TNBC but higher hepatic accumulation with a higher dose.Fig. S10. Representative characterisation of St-PGA-DOX and OximUNO.Fig. S11. LC-MS method development for the determination of DOX in drug release studies and stability studies of OximUNO in i.p. fluid and dPBS.Fig. S12. OximUNO shows no signs of hepatic or renal toxicity.Fig. S13. H&E on healthy Balb/c mouse lung.Fig. S14. Localisation of St-PGA-OG/St-PGA-OG-mUNO and tumour vessels.Fig. S15. H&E staining of hearts from the treatment of orthotopic TNBC.Fig. S16. A repetition of OximUNO treatment on the orthotopic TNBC monitoring tumour growth and survival.Fig. S17. Flow cytometry plots for M2 and M1 macrophages.Fig. S18. Flow cytometry gating for cytotoxic T lymphocytes (CTLs).Fig. S19. Flow cytometry gating for T regulatory cells (Tregs). Fig. S20. Flow cytometry analysis showing total cells.Fig. S21. Treatment with OximUNO does not affect splenic macrophages.

Supplementary Movie SM5. Tumor cell migration on plain silver nanoparticles

Supplementary Figures 1-7. Supplemental Figure 1: Metastatic burden and primary tumor weight after long term in vivo peptide treatment Supplemental Figure 2: In vitro and in vivo targeting of pancreatic tumor cells by iRGD and iNGR Supplemental Figure 3: Inhibition of tumor cell attachment to vitronectin by RGD peptides Supplemental Figure 4: In vitro cell toxicity of CendR peptides Supplemental Figure 5: Chemorepulsion assay design Supplemental Figure 6: Inhibition of NRP-1-dependent GFP-PC-3 cell attachment to fibronectin Supplemental Figure 7: Effects of CendR peptides on tumor cell attachment to collagen type-1 Legends of Supplementary Movies SM1 through SM5

Peritoneal carcinomatosis is present in more than 60% of gastric cancer, 40% of ovarian cancer, and 35% of colon cancer patients. It is the second most common cause of cancer-related mortality, with a median survival of 1 to 3 months. Cytoreductive surgery combined with intraperitoneal chemotherapy is the current clinical treatment, but achieving curative drug accumulation and penetration in peritoneal carcinomatosis lesions remains an unresolved challenge. Here, we used flexible and pH-sensitive polymersomes for payload delivery to peritoneal gastric (MKN-45P) and colon (CT26) carcinoma in mice. Polymersomes were loaded with paclitaxel and in vitro drug release was studied as a function of pH and time. Paclitaxel-loaded polymersomes remained stable in aqueous solution at neutral pH for up to 4 months. In cell viability assay on cultured cancer cell lines (MKN-45P, SKOV3, CT26), paclitaxel-loaded polymersomes were more toxic than free drug or albumin-bound paclitaxel (Abraxane). Intraperitoneally administered fluorescent polymersomes accumulated in malignant lesions, and immunofluorescence revealed an intense signal inside tumors with no detectable signal in control organs. A dual targeting of tumors was observed: direct (circulation-independent) penetration, and systemic, blood vessel–associated accumulation. Finally, we evaluated preclinical antitumor efficacy of paclitaxel-polymersomes in the treatment of MKN-45P disseminated gastric carcinoma using a total dose of 7 mg/kg. Experimental therapy with paclitaxel-polymersomes improved the therapeutic index of drug over free paclitaxel and Abraxane, as evaluated by intraperitoneal tumor burden and number of metastatic nodules. Our findings underline the potential utility of the polymersome platform for delivery of drugs and imaging agents to peritoneal carcinomatosis lesions. Mol Cancer Ther; 15(4); 670–9. ©2016 AACR.

Supplemental Table 1. Summary of statistical analysis

Figure S1: Structure of the co-polymer POEGMA-PDPA; Figure S2: Quantification of paclitaxel loading into polymersomes (PS-PTX) and PTX release from the vesicles; Figure S3: z-potential measurements; Figure S4: In vitro cellular internalization and drug release of polymersomes; Figure S5: Viability and proliferation and of MKN-45P and CT-26 cells; Figure S6: Fluorescence in control organs; Figure S7: In vivo imaging of rhodamine-labeled polymersomes; Figure S8: Homing of fluorescein-labeled polymersomes to organs; Figure S9: Subcutaneous tumor homing of polymersomes; Figure S10: Peritoneal tumor homing of polymersomes after IP and IV injection.

PDF file - 4704K, Figure S4. Tumor volume and body weight of mice bearing orthotopic 4T1 tumors and treated with intravenously injected Bit1 cell death domain constructs.

PDF file - 404K, iNGR does not bind to CD13+ monocytes

PDF file - 7120K, Figure S2. N-terminal fragments of Bit1 cause cell death.

PDF file - 382K, Peptide description. ID and sequence in single letter amino acid code of each peptide is shown

PDF file - 92K

PDF file - 166K, HUVECs express NGR and CendR motif receptors

PDF file - 2447K, Figure S3. Caspase cascade signaling is impaired in MCF-10CA1a human breast cancer cells.

PDF file - 590K, iNGR penetrates into extravascular tumor tissue

PDF file - 898K, iNGR does not increase the penetration of co-administered Evans blue dye into normal organs

PDF file - 372K, iNGR enhances tumor penetration of Evans Blue dye

PDF file - 1MB, iNGR is cleaved upon entering the cells

PDF file - 3336K, Figure S1. Construction and identification of recombinant proteins from Bit1 cell death domain.

PDF file - 517K, iNGR binding is not mediated by alphav integrins

Poor penetration of antitumor drugs into the extravascular tumor tissue is often a major factor limiting the efficacy of cancer treatments. Our group has recently described a strategy to enhance tumor penetration of chemotherapeutic drugs through use of iRGD peptide (CRGDK/RGPDC). This peptide comprises two sequence motifs: RGD, which binds to αvβ3/5 integrins on tumor endothelia and tumor cells, and a cryptic CendR motif (R/KXXR/K-OH). Once integrin binding has brought iRGD to the tumor, the peptide is proteolytically cleaved to expose the cryptic CendR motif. The truncated peptide loses affinity for its primary receptor and binds to neuropilin-1, activating a tissue penetration pathway that delivers the peptide along with attached or co-administered payload into the tumor mass. Here, we describe the design of a new tumor-penetrating peptide based on the current knowledge of homing sequences and internalizing receptors. The tumor-homing motif in the new peptide is the NGR sequence, which binds to endothelial CD13. The NGR sequence was placed in the context of a CendR motif (RNGR), and this sequence was embedded in the iRGD framework. The resulting peptide (CRNGRGPDC, iNGR) homed to tumor vessels and penetrated into tumor tissue more effectively than the standard NGR peptide. iNGR induced greater tumor penetration of coupled nanoparticles and co-administered compounds than NGR. Doxorubicin given together with iNGR was significantly more efficacious than the drug alone. These results show that a tumor-specific, tissue-penetrating peptide can be constructed from known sequence elements. This principle may be useful in designing tissue-penetrating peptides for other diseases. Cancer Res; 73(2); 804–12. ©2012 AACR.

Novel anticancer compounds and their precision delivery systems are actively developed to create potent and well‐tolerated anticancer therapeutics. Here, we report the synthesis of a novel anthracycline, Utorubicin (UTO), and its preclinical development as an anticancer payload for nanocarriers. Free UTO was significantly more toxic to cultured tumor cell lines than the clinically used anthracycline, doxorubicin. Nanoformulated UTO, encapsulated in polymeric nanovesicles (polymersomes, PS), reduced the viability of cultured malignant cells and this effect was potentiated by functionalization with a tumor‐penetrating peptide (TPP). Systemic peptide‐guided PS showed preferential accumulation in triple‐negative breast tumor xenografts implanted in mice. At the same systemic UTO dose, the highest UTO accumulation in tumor tissue was seen for the TPP‐targeted PS, followed by nontargeted PS, and free doxorubicin. Our study suggests potential applications for UTO in the treatment of malignant diseases and encourages further preclinical and clinical studies on UTO as a nanocarrier payload for precision cancer therapy., Utorubicin (UTO) is a novel anthracycline with higher anticancer activity than doxorubicin in cultured cancer cells. Nanoencapsulation of UTO in polymeric vesicles functionalized with tumor‐penetrating peptides increases the cytotoxicity of the drug in receptor‐positive cells in vitro and, after systemic administration into mice bearing triple‐negative tumors, potentiates UTO accumulation in malignant tissue.

Glioblastoma is considered one of the most aggressive malignancies in adult and pediatric patients. Despite decades of research no curative treatment is available and it thus remains associated with a very dismal prognosis. Although recent pre-clinical and clinical studies have demonstrated the feasibility of chimeric antigen receptors (CAR) T cell immunotherapeutic approach in glioblastoma, tumor heterogeneity and antigen loss remain among one of the most important challenges to be addressed. In this study, we identify p32/gC1qR/HABP/C1qBP to be specifically expressed on the surface of glioma cells, making it a suitable tumor associated antigen for redirected CAR T cell therapy. We generate p32 CAR T cells and find them to recognize and specifically eliminate p32 expressing glioma cells and tumor derived endothelial cells in vitro and to control tumor growth in orthotopic syngeneic and xenograft mouse models. Thus, p32 CAR T cells may serve as a therapeutic option for glioblastoma patients., Chimeric antigen receptor (CAR) T cell therapy has been proposed as a promising approach for treating glioblastoma. Here the authors show that p32 is expressed in murine and human glioma and that p32-directed CAR-T cells promote anti-tumor responses in preclinical models by targeting glioma cells and tumor derived endothelial cells.

Targeted therapies have increased the treatment options for triple-negative breast cancer patients. However, the paucity of targetable biomarkers and tumour heterogeneity have limited the ability of precision-guided interventions to live up to their full potential. As affinity targeting ligands, aptamers show high selectivity towards target molecules. Compared to antibodies, aptamers have lower molecular weight, increased stability during transportation, reduced immunogenicity, and increased tissue uptake. Recently, we reported the discovery of GreenB1 aptamer that is internalized in cultured triple-negative MDA-MB-231 human breast cancer cells. We show that the GreenB1 aptamer specifically targets β1-integrin, a protein previously linked to breast cancer cell invasiveness and migration. Aptamer binds to β1-integrin with low nanomolar affinity. GreenB1 homes in the orthotopic 4T1 triple-negative breast cancer lesions modelled in mice. Our findings suggest potential applications for the GreenB1-guided precision agents for the diagnosis and therapy of triple-negative breast cancer.

The present study investigated the effect of plasma-produced reactive oxygen (ROS) and nitrogen (RNS) species on cancer cell viability. Reactive species were generated in deionized water by using an atmospheric pressure Ar plasma jet within the controlled ambient gases (air, O2 or N2), which allowed the production of plasma-activated water containing only ROS (e.g. O3, H2O2) or both ROS and RNS (e.g. H2O2, NO2–, NO3–). A considerable amount of H2O2 was produced in all ambient gases, and its generation rate was highest in N2 and lowest in O2. The latter was connected with a H2O2 precursor, OH, efficient quenching in O2 ambient gas. Small quantities of NO2– were generated during short (

Background: Conjugation to clinical-grade tumor penetrating iRGD peptide is a widely used strategy to improve tumor homing, extravasation, and penetration of cancer drugs and tumor imaging agents. The C domain of the extracellular matrix molecule Tenascin-C (TNC-C) is upregulated in solid tumors and represents an attractive target for clinical-grade single-chain antibody- based vehicles for tumor delivery drugs and imaging agents. Objective: To study the effect of C-terminal genetic fusion of the iRGD peptide to recombinant anti- TNC-C single-chain antibody clone G11 on systemic tumor homing and extravasation. Methods: Enzyme-linked immunosorbent assay was used to study the interaction of parental and iRGD-fused anti-TNC-C single-chain antibodies with C domain of tenascin-C and αVβ3 integrins. For systemic homing studies, fluorescein-labeled ScFV G11-iRGD and ScFV G11 antibodies were administered in U87-MG glioblastoma xenograft mice, and their biodistribution was studied by confocal imaging of tissue sections stained with markers of blood vessels and Tenascin C immunoreactivity. Results: In a cell-free system, iRGD fusion to ScFV G11 conferred the antibody has a robust ability to bind αVβ3 integrins. The fluorescein labeling of ScFV G11-iRGD did not affect its target binding activity. In U87-MG mice, iRGD fusion to ScFV G11 antibodies improved their homing to tumor blood vessels, extravasation, and penetration of tumor parenchyma. Conclusion: The genetic fusion of iRGD tumor penetrating peptide to non-internalizing affinity targeting ligands may improve their tumor tropism and parenchymal penetration for more efficient delivery of imaging and therapeutic agents into solid tumor lesions.

M2-like tumor-associated macrophages (M2 TAMs) play important roles in the resistance of tumors to immunotherapies. Selective depletion or reprogramming of M2 TAMs may sensitize the nonresponsive tumors for immune-mediated eradication. However, precision delivery of payloads to M2 TAMs, while sparing healthy tissues, has remained an unresolved challenge. Here, we studied the application of a short linear peptide (CSPGAK, "mUNO") for the delivery of molecular and nanoscale cargoes in M2 TAMs in vitro and the relevance of the peptide for in vivo targeting of early-stage primary breast tumors and metastatic lung foci. First, we performed in silico modeling and found that mUNO interacts with mouse CD206 via a binding site between lectin domains CTLD1 and CTLD2, the same site previously demonstrated to be involved in mUNO binding to human CD206. Second, we showed that cultured M2 macrophages take up fluorescein-labeled (FAM) polymersomes conjugated with mUNO using the sulfhydryl group of its N-terminal cysteine. Pulse/chase studies of FAM-mUNO in M2 macrophages suggested that the peptide avoided lysosomal entrapment and escaped from early endosomes. Third, our in vivo studies with FAM-mUNO demonstrated that intraperitoneal administration results in better pharmacokinetics and higher blood bioavailability than can be achieved with intravenous administration. Intraperitoneal FAM-mUNO, but not FAM-control, showed a robust accumulation in M2-skewed macrophages in mouse models of early primary breast tumor and lung metastasis. This targeting was specific, as no uptake was observed in nonmalignant control organs, including the liver, or other cell types in the tumor, including M1 macrophages. Collectively, our studies support the application of the CD206-binding mUNO peptide for delivery of molecular and nanoscale cargoes to M2 macrophages and manifest the relevance of this mode of targeting primary and metastatic breast tumors.

Homing peptides and cell-penetrating peptides allow for systemic targeting of diseased tissues and/or efficient intracellular delivery of payloads [...]

Although many studies have explored the depletion of tumor-associated macrophages (TAM) as a therapeutic strategy for solid tumors, currently available compounds suffer from poor efficacy and dose-limiting side effects. Here, we developed a novel TAM-depleting agent (“OximUNO”) that specifically targets CD206+ TAMs and demonstrated efficacy in a triple-negative breast cancer (TNBC) mouse model. OximUNO comprises a star-shaped polyglutamate (St-PGA) decorated with the CD206-targeting peptide mUNO that carries the chemotherapeutic drug doxorubicin (DOX). In the TNBC model, a fluorescently labeled mUNO-decorated St-PGA homed to CD206+ TAMs within primary lesions and metastases. OximUNO exhibited no acute liver or kidney toxicity in vivo. Treatment with OximUNO reduced the progression of primary tumor lesions and pulmonary metastases, significantly diminished the number of CD206+ TAMs and increased the CD8/FOXP3 expression ratio (indicating immunomodulation). Our findings suggest the potential benefit of OximUNO as a TAM-depleting agent for TNBC treatment. Importantly, our studies also represent a novel design of a peptide-targeted St-PGA as a targeted therapeutic nanoconjugate. Significance: A peptide-targeted nanoformulation of DOX exclusively eliminates mannose receptor+ TAMs in breast cancer models, generating response without off-target effects (a drawback of many TAM-depleting agents under clinical study).

Endosomal sorting maintains cellular homeostasis by recycling transmembrane proteins and associated proteins and lipids (termed “cargoes”) from the endosomal network to multiple subcellular destinations, including retrograde traffic to the trans-Golgi network (TGN). Viral and bacterial pathogens subvert retrograde trafficking machinery to facilitate infectivity. Here, we develop a proteomic screen to identify retrograde cargo proteins of the endosomal SNX-BAR sorting complex promoting exit 1 (ESCPE-1). Using this methodology, we identify Neuropilin-1 (NRP1), a recently characterized host factor for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, as a cargo directly bound and trafficked by ESCPE-1. ESCPE-1 mediates retrograde trafficking of engineered nanoparticles functionalized with the NRP1-interacting peptide of the SARS-CoV-2 spike (S) protein. CRISPR-Cas9 deletion of ESCPE-1 subunits reduces SARS-CoV-2 infection levels in cell culture. ESCPE-1 sorting of NRP1 may therefore play a role in the intracellular membrane trafficking of NRP1-interacting viruses such as SARS-CoV-2., Proceedings of the National Academy of Sciences of the United States of America, 119 (25), ISSN:0027-8424, ISSN:1091-6490