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Implantable devices can be designed to release drugs to localized regions of tissue at sustained and reliable rates. Advances in polymer engineering have led to the design and development of drug-loaded implants with predictable, desirable release profiles. Biodegradable polyesters exhibit chemical, physical, and biological properties suitable for developing implants for pain management, cancer therapy, contraception, antiviral therapy, and other applications.This article reviews the use of biodegradable polyesters for drug-loaded implants by discussing the properties of commonly used polymers, techniques for implant formulation and manufacturing, mechanisms of drug release, and clinical applications of implants as drug delivery devices.Drug delivery implants are unique systems for safe and sustained drug release, providing high bioavailability and low toxicity. Depending on the implant design and tissue site of deployment, implants can offer either localized or systemic drug release. Due to the long history of use of degradable polyesters in medical devices, polyester-based implants represent an important class of controlled release technologies. Further, polyester-based implants are the largest category of drug delivery implants to reach the point of testing in humans or approval for human use.

Improving the performance of nanocarriers remains a major challenge in the clinical translation of nanomedicine. Efforts to optimize nanoparticle formulations typically rely on tuning the surface density and thickness of stealthy polymer coatings such as poly(ethylene glycol) (PEG). Here, we show that modulating the surface topography of PEGylated nanoparticles using bottlebrush block copolymer (BBCP) significantly enhances circulation and tumor accumulation providing an alternative strategy to improve nanoparticle coatings. Specifically, nanoparticles with rough surface topography achieve high tumor cell uptake in vivo due to superior tumor extravasation and distribution compared to conventional smooth-surfaced nanoparticles. Furthermore, surface topography profoundly impacts the interaction with serum proteins resulting in the adsorption of fundamentally different proteins onto the surface of rough-surfaced nanoparticles formed from BBCPs. We envision that controlling the nanoparticle surface topography of PEGylated nanoparticles will enable the design of improved nanocarriers in various biomedical applications.

Supporting information include 3 tables and 3 figures. Table 1 describes the Patients characteristics from which primary cell lines were established. Table 2 the characteristics of ovarian tumor samples from which RNA was extracted. Table 3 the characterization of particle formulations Supplementary figure 1 shows the DNA release in vitro in culture medium. The Supplementary figure 2 the ovarian tumors' uptake of c-CPE-NP in vivo. The Supplementary figure 3 shows the survival curves of the tumor bearing mice treated with vehicle, c-CPE-NP encapsulating the empty plasmid or the p16 DTA c-CPE-NP.

Fig S2. Cell growth of bladder cancer cells upon SC144 treatment.

Supplementary Figure 1. Encapsulation efficiency (drug loading/theoretical drug encapsulated) of candidate drugs into PLA-PEG NPs as a function of their solubility in DMSO; Supplementary Figure 2. PLA-PEG NP in vitro stability and uptake; Supplementary Figure 3. in vitro dose-dependent response of HR (L) and mNHEJ (R) pathway repair in the U2OS reporter assay to treatment with either free drug (red) or drug-NP (green) for each candidate radiosentizier; Supplementary Figure 4. dsDNA repair pathway activity levels in U2OS reporter cells; Supplementary Table 1. Results of Linear Regression Analysis and Correlation testing between drug properties and release properties; Supplementary Table 2. Summary of NP-drug formulations and established criteria for candidate drug-NP formulation selection.

Results of the Lung Cancer Cell Line Screen for OST Inhibitor Sensitivity

Purpose:Parallel signaling reduces the effects of receptor tyrosine kinase (RTK)–targeted therapies in glioma. We hypothesized that inhibition of protein N-linked glycosylation, an endoplasmic reticulum co- and posttranslational modification crucial for RTK maturation and activation, could provide a new therapeutic approach for glioma radiosensitization.Experimental Design: We investigated the effects of a small-molecule inhibitor of the oligosaccharyltransferase (NGI-1) on EGFR family receptors, MET, PDGFR, and FGFR1. The influence of glycosylation state on tumor cell radiosensitivity, chemotherapy-induced cell toxicity, DNA damage, and cell-cycle arrest were determined and correlated with glioma cell receptor expression profiles. The effects of NGI-1 on xenograft tumor growth were tested using a nanoparticle formulation validated by in vivo molecular imaging. A mechanistic role for RTK signaling was evaluated through the expression of a glycosylation-independent CD8-EGFR chimera.Results:NGI-1 reduced glycosylation, protein levels, and activation of most RTKs. NGI-1 also enhanced the radiosensitivity and cytotoxic effects of chemotherapy in those glioma cells with elevated ErbB family activation, but not in cells without high levels of RTK activation. NGI-1 radiosensitization was associated with increases in both DNA damage and G1 cell-cycle arrest. Combined treatment of glioma xenografts with fractionated radiotherapy and NGI-1 significantly reduced tumor growth compared with controls. Expression of the CD8-EGFR eliminated the effects of NGI-1 on G1 arrest, DNA damage, and cellular radiosensitivity, identifying RTK inhibition as the principal mechanism for the NGI-1 effect.Conclusions:This study suggests that oligosaccharyltransferase inhibition with NGI-1 is a novel approach to radiosensitize malignant gliomas with enhanced RTK signaling.See related commentary by Wahl and Lawrence, p. 455

Deep sequencing of lung cancer cell lines to determine the proportion of EGFR mutations.

Time course resolution of γH2AX foci formation in D54 cells

List of antibodies used in the study

Supplementary figures legends

Characterization of the Osimertinib resistant H1975 cells.

Legends for supplementary figures 1 and 2

Dose response of NGI-1, temozolomide and etoposide in glioma cell lines

Asparagine (N)-linked glycosylation is a posttranslational modification essential for the function of complex transmembrane proteins. However, targeting glycosylation for cancer therapy has not been feasible due to generalized effects on all glycoproteins. Here, we perform sensitivity screening of 94 lung cancer cell lines using NGI-1, a small-molecule inhibitor of the oligosaccharyltransferase (OST) that partially disrupts N-linked glycosylation, and demonstrate a selective loss of tumor cell viability. This screen revealed NGI-1 sensitivity in just 11 of 94 (12%) cell lines, with a significant correlation between OST and EGFR inhibitors. In EGFR-mutant non-small cell lung cancer with EGFR tyrosine kinase inhibitor (TKI) resistance (PC9-GR, HCC827-GR, and H1975-OR), OST inhibition maintained its ability to induce cell-cycle arrest and a proliferative block. Addition of NGI-1 to EGFR TKI treatment was synthetic lethal in cells resistant to gefitinib, erlotinib, or osimertinib. OST inhibition invariably disrupted EGFR N-linked glycosylation and reduced activation of receptors either with or without the T790M TKI resistance mutation. OST inhibition also dissociated EGFR signaling from other coexpressed receptors like MET via altered receptor compartmentalization. Translation of this approach to preclinical models was accomplished through synthesis and delivery of NGI-1 nanoparticles, confirmation of in vivo activity through molecular imaging, and demonstration of significant tumor growth delay in TKI-resistant HCC827 and H1975 xenografts. This therapeutic strategy breaks from kinase-targeted approaches and validates N-linked glycosylation as an effective target in tumors driven by glycoprotein signaling.Significance: EGFR-mutant NSCLC is incurable despite the marked sensitivity of these tumors to EGFR TKIs. These findings identify N-linked glycosylation, a posttranslational modification common to EGFR and other oncogenic signaling proteins, as an effective therapeutic target that enhances tumor responses for EGFR-mutant NSCLC. Cancer Res; 78(17); 5094–106. ©2018 AACR.

Figure S3, Related to Figure 4. A, c-SRC expression in normal (HFF and MCF-10A), luminal-A (MCF-7), LAR-TNBC (MDA-MB-453), and mesenchymal-TNBC (MDA-MB-231, BT-549, and Hs578T) cell lines. Expression was normalized to GAPDH and made relative to c-SRC expression in HFF lines. B-C, Clonogenic assays in Hs578T (B), and MDA-MB-436 (C) TNBC cells after miR-34a transfection and treatment with Dasatnib (left panels) and paclitaxel (right panel) or as a control HeLa cells after dasatinib treatment (C, right panel). D-E, Assessment of miR-34a target genes in MDA-MB-231 cells after 72 hours of dasatinib treatment (D), or miR-34a levels after 72 hours paclitaxel treatment (E). F, miR-34a promoter luciferase assays in MDA-MB-31 cells after dasatinib treatment. G-H, Spearman rank correlation analysis of SRC levels in cell lines described in Figure 1A (G), and in all breast cancer samples in the Metabric dataset (H). I, Comparable decreases in phospho-Tyr416 active c-SRC, non-phospho-Tyr527 c-SRC, and total c-SRC are observed in MDA-MB-231 cells transfected with 15nM miR-34a versus miR-Scr control, as determined by Western blot analysis. J, Schematic highlighting the miR-34a-c-SRC double-negative feedback loop present in MSL TNBC cells, which can be influenced by exogenous addition of miR-34a, or by dasatinib treatment. * Indicates p

Figure S1, Related to Figure 1. A, miRNA microarray data of the top 50 most variant miRNAs across 17 breast cancer cell lines representing either basal/TNBC (top left blue bar) or luminal (top right red bar) breast cancer subgroups. Amongst these miRNAs, miR-34a (red asterisk) was found to be uniquely downregulated in basal lines. B-C, qPCR validation of array data in TNBC and Luminal-A cancer cell lines as compared to normal mammary epithelial lines (CRCs were cultured using the ROCK inhibitor and condition medium from the 3T3 feeder system as previously described(20)). D, Schematic of WebGestalt 2 analysis of Affymetrix microarray data across 19 cell lines. ¬¬miR-34a is one of several miRNAs with target enrichment in the TNBC overexpressed gene set. E, Results of hypergeometric analysis of miR-34a targets using a more stringent context score cutoff of -0.27. F, SRB growth assays on additional TNBC and normal cell lines.

Figure S4, Related to Figure 5. A-F, Further characterization of c-SRC siRNA treatments in TNBC cell lines. A, Crystal violet staining of MDA-MB-231 and Hs578T cells post 15nM si-SRC and si-Neg transfection (Day 6 images, left panel), and quantification of staining abundance is shown on right panels). B, SRB results on Hs578T cell line. C, Analysis of a second siRNA to c-SRC in MDA-MB-231 cells. SRB assays are shown in the left panel and western blot analysis confirming c-SRC knockdown (72 hour post-transfection) is shown on the right panel. D, Crystal violet staining of HFF cells. E, SA-β-gal assays in the indicated cell lines 5 days after 15nM si-SRC transfection. F, miR-34a levels 72 hours after 15nM si-SRC transfection in Hs578T cells. G, Assessment of SRC mRNA levels in the indicated cell lines 72 hours after 15nM si-SRC transfection.

Triple-negative breast cancer (TNBC) is an aggressive subtype with no clinically proven biologically targeted treatment options. The molecular heterogeneity of TNBC and lack of high frequency driver mutations other than TP53 have hindered the development of new and effective therapies that significantly improve patient outcomes. miRNAs, global regulators of survival and proliferation pathways important in tumor development and maintenance, are becoming promising therapeutic agents. We performed miRNA-profiling studies in different TNBC subtypes to identify miRNAs that significantly contribute to disease progression. We found that miR-34a was lost in TNBC, specifically within mesenchymal and mesenchymal stem cell–like subtypes, whereas expression of miR-34a targets was significantly enriched. Furthermore, restoration of miR-34a in cell lines representing these subtypes inhibited proliferation and invasion, activated senescence, and promoted sensitivity to dasatinib by targeting the proto-oncogene c-SRC. Notably, SRC depletion in TNBC cell lines phenocopied the effects of miR-34a reintroduction, whereas SRC overexpression rescued the antitumorigenic properties mediated by miR-34a. miR-34a levels also increased when cells were treated with c-SRC inhibitors, suggesting a negative feedback exists between miR-34a and c-SRC. Moreover, miR-34a administration significantly delayed tumor growth of subcutaneously and orthotopically implanted tumors in nude mice, and was accompanied by c-SRC downregulation. Finally, we found that miR-34a and SRC levels were inversely correlated in human tumor specimens. Together, our results demonstrate that miR-34a exerts potent antitumorigenic effects in vitro and in vivo and suggests that miR-34a replacement therapy, which is currently being tested in human clinical trials, represents a promising therapeutic strategy for TNBC. Cancer Res; 76(4); 927–39. ©2015 AACR.

Figure S5, Related to Figure 6. A, Assessment of SRC mRNA levels in MDA-MB-231 SRC-ORF, EMPTY-ORF, or parental cell lines. Data is normalized in RPL19 levels. B, Crystal violet staining assays on day 5 post-transfection confirms the ability of c-SRC to rescue miR-34a-induced anti-tumor growth. C-D, Examples of Pearson correlation analysis indicating MDA-MB-231 cells were not similar to BT-549 and MDA-MB-436 cells, and therefore were not included in the initial K-Means clustering analysis (results shown in D). E, The fold knockdown by miR-34a as compared to the miR-Scr treatments of the indicated genes in Clusters 1-3 in both BT-549 and MDA-MB-436 cells using a 2-fold change cut-off. None of the genes in Cluster 4 were downregulated by miR-34a (data not shown). F, Schematic of the KEGG pathway (hsa04510: Focal Adhesion) with miR-34a downregulated genes highlighted in red. G-H, Represents further analysis of the miR-34a gene signature in breast cancer. G, Indicates correlation analyses of miR-34a target genes in TNBC patients from Metabric data. H, Confirmation of prognostic importance of mIR-34a gene signature using a PROGgeneV2 algorithm on the TCGA data set.

Supplementary Materials and Methods and Supplementary Figure Legends

Figure S2, Related to Figure 2. A-F, Functional characterization of miR-34a re-introduction in additional TNBC and non-TNBC cell lines by way of Matrigel-invasion (A), soft agar growth (B), and BrdU labeling (C) experiments. For BrdU assays, we observed only a 5-10% accumulation of BrdU-positive cells 4 days post-transfection. SA-β-gal assays on MDA-MB-157 (D), and BT-20 (E, left panel) TNBC cells, as compared to MCF-7 luminal-A cells (E, right panel). F, MCF-7 cells stably transfected with either a miR-34a (miR-34a SP) or a control (Empty SP) reporter/sponge construct was assayed for SA-β-gal activity. Two independent miR-34a SP pools were generated. G, Luciferase reporter assays indicative of miR-34a activity in all TNBC, luminal, and normal cell lines used in this study. H, miRNA levels in MDA-MB-231, MDA-MB-436, and BT-549 TNBC cells or BT-474 and MCF-7 Luminal-A cells 72 hours after 10nM miR-34a or miR-Scr transfection. In TNBC cells miR-34a expressing lines harbored lower levels of miR-17/92 family members as compared to miR-Scr treated lines. * Indicates p

Myelomeningocele (MMC) is a devastating congenital neurologic disorder that can lead to lifelong morbidity and has limited treatment options. This study investigates the use of poly(lactic-co-glycolic acid) (PLGA) microparticles (MPs) loaded with fibroblast growth factor (FGF) as a platform for in utero treatment of MMC.Intra-amniotic injections of PLGA MPs were performed on gestational day 17 (E17) in all-trans retinoic acid-induced MMC rat dams. MPs loaded with fluorescent dye (DiO) were evaluated 3 hours after injection to determine incidence of binding to the MMC defect. Fetuses were then treated with PBS or PLGA particles loaded with DiO, bovine serum albumin, or FGF and evaluated at term (E21). Fetuses with MMC defects were evaluated for gross and histologic evidence of soft tissue coverage. The effect of PLGA-FGF treatment on spinal cord cell death was evaluated using an in situ cell death kit.PLGA-DiO MPs had a binding incidence of 86% and 94% 3 hours after injection at E17 for doses of 0.1 mg and 1.2 mg, respectively. Incidence of soft tissue coverage at term was 19% (4 of 21), 22% (2 of 9), and 83% (5 of 6) for PLGA-DiO, PLGA-BSA, and PLGA-FGF, respectively. At E21, the percentage of spinal cord cells positive for in situ cell death was significantly higher in MMC controls compared with wild-type controls or MMC pups treated with PLGA-FGF.PLGA MPs are an innovative minimally invasive platform for induction of soft tissue coverage in the rat model of MMC and may reduce cellular apoptosis.

Glioblastoma (GBM) is one of the most lethal malignancies with poor survival and high recurrence rates. Here, we aimed to simultaneously target oncomiRs 10b and 21, reported to drive GBM progression and invasiveness. We designed short (8-mer) γ-modified peptide nucleic acids (sγPNAs), targeting the seed region of oncomiRs 10b and 21. We entrapped these anti-miR sγPNAs in nanoparticles (NPs) formed from a block copolymer of poly(lactic acid) and hyperbranched polyglycerol (PLA-HPG). The surface of the NPs was functionalized with aldehydes to produce bioadhesive NPs (BNPs) with superior transfection efficiency and tropism for tumor cells. When combined with temozolomide, sγPNA BNPs administered via convection-enhanced delivery (CED) markedly increased the survival (>120 days) of two orthotopic (intracranial) mouse models of GBM. Hence, we established that BNPs loaded with anti-seed sγPNAs targeting multiple oncomiRs are a promising approach to improve the treatment of GBM, with a potential to personalize treatment based on tumor-specific oncomiRs.

Gene amplification drives oncogenesis in a broad spectrum of cancers. A number of drugs have been developed to inhibit the protein products of amplified driver genes, but their clinical efficacy is often hampered by drug resistance. Here, we introduce a therapeutic strategy for targeting cancer-associated gene amplifications by activating the DNA damage response with triplex-forming oligonucleotides (TFOs), which drive the induction of apoptosis in tumors, whereas cells without amplifications process lower levels of DNA damage. Focusing on cancers driven by HER2 amplification, we find that TFOs targeting HER2 induce copy number-dependent DNA double-strand breaks (DSBs) and activate p53-independent apoptosis in HER2-positive cancer cells and human tumor xenografts via a mechanism that is independent of HER2 cellular function. This strategy has demonstrated in vivo efficacy comparable to that of current precision medicines and provided a feasible alternative to combat drug resistance in HER2-positive breast and ovarian cancer models. These findings offer a general strategy for targeting tumors with amplified genomic loci. Amplified loci in cancer genomes are directly targeted with triplex-forming oligonucleotides.

Peptide nucleic acids (PNAs), a synthetic DNA mimic, have been extensively utilized for antisense- and antigene-based biomedical applications. Significant efforts have been made to increase the cellular uptake of PNAs, but here we examined relatively unexplored aspects of intracellular trafficking and endocytic recycling of PNAs. For proof-of-concept, we used anti-microRNA (miR) PNA targeting miR-155. The sub-cellular localization of PNA was studied via confocal and flow-cytometry-based assays in HeLa cells. A comprehensive characterization of PNA-containing extracellular vesicles revealed spherical morphology, negative surface charge density, and the presence of tetraspanin markers. Most importantly, we investigated rab11a and rab27b GTPases’ role in regulating the exocytosis of PNAs. Organelle staining, followed by confocal imaging, showed higher localization of PNA in lysosomes. Gene-expression analysis established the enhanced functional activity of PNA after inhibition of endocytic recycling. Multiple studies report the exocytosis of single-stranded oligonucleotides, short interfering RNAs (siRNAs), and nanocarriers. To our knowledge, this is the first mechanistic study to establish that PNA undergoes endocytic recycling and exocytosis out of tumor cells. The results presented here can serve as a platform to develop and optimize strategies for improving the therapeutic efficacy of PNAs by avoiding the recycling pathways., Graphical abstract, Authors investigated that peptide nucleic acid undergoes exocytosis in the cancer cells in a series of cell-culture-based functional assays. It was noted that membrane trafficking proteins play a significant role in the exocytosis and intracellular trafficking of peptide nucleic acid.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic has highlighted the need for vaccines that not only prevent disease but also prevent transmission. Parenteral vaccines induce robust systemic immunity but poor immunity at the respiratory mucosa. We developed a vaccine strategy that we call “prime and spike,” which leverages existing immunity generated by primary vaccination (prime) to elicit mucosal immune memory within the respiratory tract by using unadjuvanted intranasal spike boosters (spike). We show that prime and spike induces robust resident memory B and T cell responses, induces immunoglobulin A at the respiratory mucosa, boosts systemic immunity, and completely protects mice with partial immunity from lethal SARS-CoV-2 infection. Using divergent spike proteins, prime and spike enables the induction of cross-reactive immunity against sarbecoviruses.

This Review examines the state-of-the-art in the delivery of nucleic acid therapies that are directed to the vascular endothelium. First, we review the most important homeostatic functions and properties of the vascular endothelium and summarize the nucleic acid tools that are currently available for gene therapy and nucleic acid delivery. Second, we consider the opportunities available with the endothelium as a therapeutic target and the experimental models that exist to evaluate the potential of those opportunities. Finally, we review the progress to date from investigations that are directly targeting the vascular endothelium: for vascular disease, for peri-transplant therapy, for angiogenic therapies, for pulmonary endothelial disease, and for the blood-brain barrier, ending with a summary of the future outlook in this field.