Primary Central Nervous System Lymphoma is an aggressive central nervous system neoplasm with poor response to pharmacological treatment, partially due to insufficient drug delivery across blood-brain barrier. In this study, we developed a novel therapy for this lymphoma by combining a targeted nanopolymer treatment with an immune checkpoint inhibitor antibody (anti-PD-1). A N-(2-hydroxypropyl)methacrylamide copolymer-based nanoconjugate was designed to block tumor cell c-Myc oncogene expression by antisense oligonucleotide. Angiopep-2 peptide was conjugated to the copolymer to facilitate nanodrug crossing of the blood-brain barrier. Systemically administered polymeric nanodrug, alone or in combination with immune checkpoint inhibitor antibody anti-PD-1, was tested in syngeneic mouse model of A20 intracranial brain lymphoma. There was no significant survival difference between saline- and free anti-PD-1-treated groups. However, significant survival advantage vs. saline was observed upon treatment with nanodrug bearing Angiopep-2, H6 (6 histidines for endosome escape), and c-Myc antisense alone and especially when it was combined with anti-PD-1 antibody. Animal survival after combined treatment was also significantly increased vs. free anti-PD-1. Artificial Intelligence-assisted analysis of gene expression database after RNA-seq of tumors was used to find novel immune pathways, molecular targets and the most effective multifunctional drugs together with future drug prediction for brain lymphoma in vivo model. Spectral flow cytometry and RNA-seq analysis revealed a robust activation of tumor infiltrating T lymphocytes with enhanced interferon γ signaling and polarization to M1-type macrophages in treated tumors, which was confirmed by immunofluorescence staining. In summary, a new effective blood-brain barrier crossing nano immuno therapeutic system was developed that effectively blocked tumor c-Myc acting in combination with immune checkpoint inhibitor anti-PD-1 to treat primary brain lymphoma. The treatment improved survival of tumor-bearing animals through activation of both the adaptive and innate immune responses.

Glioblastomas (GBM) escape the immune system and are heterogenous making it difficult to target the tumor directly for treatment. However, tumor microenvironment is independent of glioma heterogeneity and might be a universal target. GBMs overexpressing laminin-411 are associated with poor patient survival. We synthesized a blood brain barrier (BBB) crossing drug delivery system that can selectively inhibit laminin-411 synthesis in vivo, upregulating innate immune system and improving survival. GBM cell lines CT-2A and GL261 with CRISPR/Cas9 depletion of laminin-411 or control wild-type were intracranially implanted in C57BL/6J mice, n = 8 per group. Depletion was validated by western blot. We used flow cytometry (FC) to verify molecular changes in tumor microenvironment after laminin-411 depletion and examined mouse survival. Additionally, wild-type GL261-implanted mice (n=8) were I.V. treated with nanoimmuno polymeric drug crossing BBB and specifically blocking laminin-411 synthesis using morpholino antisense. Mouse survival was determined, and tumors were assessed by RNA-seq, immune markers (FC), and immunohistochemistry. Animals with laminin-411 knockout GBMs showed significant tumor upregulation of innate immune system markers, CD3, CD8, as well as NK T cells, NK, IFNg+ NK cells, and M1 macrophages by FC analyses vs. control tumors. These mice had increased median survival, 47 vs. 34 days in CT-2A (p = 0.003) and over 50 vs. 26 days in GL261 (p = 0.001). Similarly, in vivo treatment of wild-type GBM GL261-bearing mice with nanoimmuno polymeric drug showed 32% greater survival (33 vs. 25 days, p = 0.0001) vs. PBS. Immunohistochemistry-validated RNA-seq showed increase in IFN-g, TNF-a, and M1 macrophage iNOS, and decrease of proliferation markers Ki-67 and c-Myc after treatment. For the first time, laminin-411 was studied as a targetable regulator of GBM immunity. Our nanoimmuno drug inhibiting laminin-411 can cross BBB and inhibit tumor growth.

Glioblastoma Multiforme (GBM) represents a significant clinical challenge amongst central nervous system (CNS) tumors, with a dismal mean survival rate of less than 8 months, a statistic that has remained largely unchanged for decades (National Brain Society, 2022). The specialized intricate anatomical features of the brain, notably the blood-brain barrier (BBB), pose significant challenges to effective therapeutic interventions, limiting the potential reach of modern advancements in immunotherapy to impact these types of tumors. This study introduces an innovative, actively targeted immunotherapeutic nanoconjugate (P12/AP-2/NCs) designed to serve as an immunotherapeutic agent capable of traversing the BBB via LRP-1 receptor-mediated transcytosis. P12/AP-2/NCs exert its immune-modulating effects by inhibiting the PD-1/PD-L1 axis through a small-size PD-L1/PD-L2 antagonist peptide Aurigene NP-12 (P12). P12/AP-2/NCs are synthesized from completely biodegradable, functionalized high molecular weight β-poly(L-malic acid) (PMLA) polymer, conjugated with P12 and Angiopep-2 (AP2) to yield P12/AP-2/NCs. Evaluating nanoconjugates for BBB permeability and 3-D tumor model efficacy using an in vitro BBB-Transwell spheroid based model demonstrating successful crossing of the BBB and internalization in brain 3D tumor environments. In addition, the nanoconjugate mediated T cell's cytotoxicity on 3D tumor region death in a U87 GBM 3-D spheroid model. AP2/P12/NCs is selectively inhibited in PD1/PDL1 interaction on T cells and tumor site, increasing inflammatory cytokine secretion and T cell proliferation. In an in-vivo murine brain environment, rhodamine fluorophore-labeled AP2/P12/NCs displayed significantly increased accumulation in the brain during 2-6 h time intervals post-injection with a prolonged bioavailability over unconjugated peptides. AP2/P12/NCs demonstrated a safety profile at both low and high doses based on major organ histopathology evaluations. Our findings introduce a novel, programmable nanoconjugate platform capable of penetrating the BBB for directed delivery of small peptides and significant immune environment modulation without utilizing antibodies, offering promise for treating challenging brain diseases like glioblastoma multiforme and beyond.

Primary central nervous system lymphoma (PCNSL) is a lethal cancer with poor survival, especially in its recurrent form. This is mainly due to low penetrance of therapeutic agents across the blood brain barrier (BBB). We created a novel nano immunodrug (nanodrug) that can cross the BBB and deliver anti-cancer agents directly to the tumor. The RNA therapeutics based on N-(2-hydroxypropyl)methacrylamide copolymer nanoplatform that was designed to block lymphoma’s cell c-Myc protein synthesis and to ensure brain tumor targeting and BBB crossing. Inhibiting of c-Myc protein synthesis played a dual role as anti-tumor proliferative factor and immune stimulator. Angiopep-2 peptide is conjugated to the nano platform to provide BBB crossing and brain lymphoma cell targeting via LRP-1 receptor. The nanodrug also contains H6 (6 histidines for endosome escape) to release into lymphoma cell cytoplasm. We tested the nanodrug in a A20 intracranial brain lymphoma mouse model alone, and in combination with anti-PD-1 antibody. Treatment with nanodrug resulted in a significant survival advantage compared to control. Survival was significantly enhanced when the nanodrug was co-injected with anti-PD-1. Spectral flow cytometry and RNA-seq analysis of treated tumors showed robust activation of tumor-infiltrating T lymphocytes with enhanced interferon γ signaling and polarization to M1-type macrophages. Artificial Intelligence-assisted analysis of gene expression data from RNA-seq revealed novel immune pathways, molecular targets, and suggested effective multifunctional drugs. Overall, we created a novel nano therapeutic drug delivery system that inhibits tumor c-Myc protein to treat PCNSL. When used in conjunction with anti-PD-1 checkpoint inhibitor, the treatment results in enhanced survival of tumor bearing animals by activating both adaptive and immune responses. NIH grants: R01 CA246716, R01 CA206220, R01 CA209921, R01 CA284247

Figures S8 and S9. Quantification of expression of laminin-411 β1 chain, its binding β1 integrin, Notch family members and other stem cell markers after nanobioconjugate treatment. All markers were significantly suppressed in treated tumors.

Figures S2, S3 and S4. Morphometric analysis of laminin-411 β1 chain (S2) and cancer stem cell markers (Notch-1, Notch-3, CD133, Nestin, and c-Myc; S3 and S4) on frozen sections of patient brain tumors. The expression of all markers significantly correlated with glioma grade

Figure S5. Binding of the nanobioconjugates to U87MG studied by flow cytometry is inhibited by human soluble TfR. This shows that the chimeric anti-human TfR antibody as part of nanobioconjugate fully retained its activity

Table S5. Nanobioconjugate toxicity test in human blood by cytokine release. No significant cytokine release was observed, compatible with lack of drug toxicity

Introduction: Tumor growth, invasion, and escape from immune surveillance largely depend on cancer microenvironment. Laminins are trimeric proteins and essential components of glioblastoma (GBM) microenvironment/extracellular matrix (ECM). In brain glioma samples from 230 patients, we found a correlation between the overexpression of tumor ECM protein laminin-411 (α4β1γ1) and faster tumor recurrence with shorter patient survival. Laminin-411 is produced by endothelial cells, neutrophils, monocytes, platelets, lymphocytes, and glioma cells and can modulate the immune system. Novel nanotechnology approach to block trimeric laminin-411 and activate of brain local immune system with brain delivery of PD-1 checkpoint inhibitor was developed for future translational application. Methods: Nanobioconjugates (NBC) based on poly (β-L-malic acid, P), were synthesized, characterized and used to intravenously treat mice with intracranial syngeneic GL261 or CT-2A GBM. The lead NBCs P/PEG/LLL(40%)/AON(α4β1)(2.0%)/AP-2(2%) and P/PEG/LLL(40%)/AP-2(2%)/αPD-1(0.2%) contained antisense to laminin-411 α4 and β1 chains, or αPD-1 antibody as well as trileucine (LLL) peptide for endosomal escape and AP-2 peptide for BBB crossing and tumor cell targeting. CRISPR/Cas9 constructs were used to knockdown α4 and β1 laminin chains in GBM ex vivo. Flow cytometry and RNA-seq analyses were performed to evaluate treatment. Results: Laminin-411 depletion with CRISPR/Cas9 and multifunctional NBC in vivo treatment equally suppressed GBM growth and significantly prolonged animal survival. The brain privileged immune system was activated upon treatment with a significant increase of CD3+, CD8+ T cells, NK, IFNγ+ NK cells, and M1 macrophages. RNA-sec analyses after treatment with a combination of NBC suppressing laminin-411 and checkpoint PD-1 showed enhanced anti-tumor effect with upregulation of genes coding for apoptotic Caspase 3 and IFNγ, and reduction of proliferation markers EGFR, c-Myc and Ki-67. Conclusion: Study describes novel GBM treatment strategy via NBCs crossing blood-brain barrier and targeting critical ECM and immune components of tumor microenvironment largely independent of heterogeneous genetic mutations in glioblastoma. Citation Format: Alexander V. Ljubimov, Rameshwar Patil, Hui Ding, Liron Israel, Eggehard Holler, Julia Y. Ljubimova, Tao Sun, Keith L. Black. Brain delivery of clinically suitable nanobioconjugates to inhibit glioblastoma growth through extracellular matrix-immune cell crosstalk [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 575.

Table S4. Physico-chemical characterization of used control and treatment nanobioconjugates

Aims/hypothesis Diabetes is associated with epigenetic modifications including DNA methylation and miRNA changes. Diabetic complications in the cornea can cause persistent epithelial defects and impaired wound healing due to limbal epithelial stem cell (LESC) dysfunction. In this study, we aimed to uncover epigenetic alterations in diabetic vs non-diabetic human limbal epithelial cells (LEC) enriched in LESC and identify new diabetic markers that can be targeted for therapy to normalise corneal epithelial wound healing and stem cell expression. Methods Human LEC were isolated, or organ-cultured corneas were obtained, from autopsy eyes from non-diabetic (59.87±20.89 years) and diabetic (71.93±9.29 years) donors. The groups were not statistically different in age. DNA was extracted from LEC for methylation analysis using Illumina Infinium 850K MethylationEPIC BeadChip and protein was extracted for Wnt phospho array analysis. Wound healing was studied using a scratch assay in LEC or 1-heptanol wounds in organ-cultured corneas. Organ-cultured corneas and LEC were transfected with WNT5A siRNA, miR-203a mimic or miR-203a inhibitor or were treated with recombinant Wnt-5a (200 ng/ml), DNA methylation inhibitor zebularine (1–20 µmol/l) or biodegradable nanobioconjugates (NBCs) based on polymalic acid scaffold containing antisense oligonucleotide (AON) to miR-203a or a control scrambled AON (15–20 µmol/l). Results There was significant differential DNA methylation between diabetic and non-diabetic LEC. WNT5A promoter was hypermethylated in diabetic LEC accompanied with markedly decreased Wnt-5a protein. Treatment of diabetic LEC and organ-cultured corneas with exogenous Wnt-5a accelerated wound healing by 1.4-fold ( p <0.05) and 37% ( p <0.05), respectively, and increased LESC and diabetic marker expression. Wnt-5a treatment in diabetic LEC increased the phosphorylation of members of the Ca 2+ -dependent non-canonical pathway (phospholipase Cγ1 and protein kinase Cβ; by 1.15-fold [ p <0.05] and 1.36-fold [ p <0.05], respectively). In diabetic LEC, zebularine treatment increased the levels of Wnt-5a by 1.37-fold ( p <0.01)and stimulated wound healing in a dose-dependent manner with a 1.6-fold (p<0.01) increase by 24 h. Moreover, zebularine also improved wound healing by 30% ( p <0.01) in diabetic organ-cultured corneas and increased LESC and diabetic marker expression. Transfection of these cells with WNT5A siRNA abrogated wound healing stimulation by zebularine, suggesting that its effect was primarily due to inhibition of WNT5A hypermethylation. Treatment of diabetic LEC and organ-cultured corneas with NBC enhanced wound healing by 1.4-fold ( p <0.01) and 23.3% ( p <0.05), respectively, with increased expression of LESC and diabetic markers. Conclusions/interpretation We provide the first account of epigenetic changes in diabetic corneas including dual inhibition of WNT5A by DNA methylation and miRNA action. Overall, Wnt-5a is a new corneal epithelial wound healing stimulator that can be targeted to improve wound healing and stem cells in the diabetic cornea. Data availability The DNA methylation dataset is available from the public GEO repository under accession no. GSE229328 ( https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE229328 ). Graphical Abstract

Tables S1, S2 and S3, and Figure S1. List of used antibodies (Table S1), and immunohistochemical expression of laminin-411 and laminin-421 in human gliomas of various grades (Tables S2 and S3). Expression of laminin-411 vs. laminin-421 increases with glioma grade and in GBMs it correlates with shorter patient survival and faster tumor recurrence (Figure S1)

Delivery of therapeutic substances into the brain poses a significant challenge in the treatment of neurological disorders. This is primarily due to the blood-brain barrier (BBB), which restricts access, alongside the limited stability and distribution of these agents within the brain tissue. Here we demonstrate an efficient delivery of microRNA (miRNA) and antisense RNA preferentially to neurons compared to astroglia in the brain of healthy and Alzheimer's disease mice, via disulfide-linked conjugation with poly(ss-L-malic acid-trileucine)-copolymer a biodegradable, amphiphilic, and multivalent platform. By conjugating a D-configured (D3)-peptide (vector) for specific targeting, highly efficient delivery across the BBB is achieved through the Low-Density Lipoprotein Receptor-Related Protein-1 (LRP-1) transcytosis pathway, amyloid beta (A13) peptides. Nanodrug distribution was determined by fluorescent labeling and analyzed by microscopy in neurons, astroglia, and in extracellular amyloid plaques typical for Alzheimer's disease. Whereas D-configured BBB-vectors can efficiently target neurons, Lconfigured (e.g., AP2-peptide) guided vector can only cross BBB but not seem to bind neurons. An analysis of post-injection fluorescence distribution, and RNA-seq followed by real-time PCR validation, confirmed a successful in vivo delivery of morpholino-miRNA-186 nanoconjugates into mouse brain. The size and fluorescence intensity of the intracellular nanodrug particulates were analyzed and verified by a competition with nonfluorescent conjugates. Differentially expressed genes (DEGs) from RNA-seq were identified in the nanodrug injected mice, and the changes of selected DEGs related to Alzheimer's disease were further validated by western blot and real-time PCR. Collectively, these results demonstrated that D3-peptide-conjugated nanopolymer drug is able to achieve neuron-selective delivery of miRNA and can serve as an efficient brain delivery vehicle in Alzheimer's disease (AD) mouse models.

Video schematically shows the mechanism of glioblastoma treatment by laminin-411 inhibiting nanobioconjugate

Figures S6 and S7. Localization of labeled nanobioconjugates in tumors and biodistribution in brain. The nano drug is seen in the tumor parenchyma outside of blood vessels (S6) and preferentially accumulates in the tumor (peaks at 6 hr post-injection) as compared to contralateral brain tissue (S7)

Air pollution poses a significant threat to human health, though a clear understanding of its mechanism remains elusive. In this study, we sought to better understand the effects of various sized particulate matter from polluted air on Alzheimer's disease (AD) development using an AD mouse model. We exposed transgenic Alzheimer's mice in their prodromic stage to different sized particulate matter (PM), with filtered clean air as control. After 3 or 6 months of exposure, mouse brains were harvested and analyzed. RNA-seq analysis showed that various PM have differential effects on the brain transcriptome, and these effects seemed to correlate with PM size. Many genes and pathways were affected after PM exposure. Among them, we found a strong activation in mRNA Nonsense Mediated Decay pathway, an inhibition in pathways related to transcription, neurogenesis and survival signaling as well as angiogenesis, and a dramatic downregulation of collagens. Although we did not detect any extracellular Aβ plaques, immunostaining revealed that both intracellular Aβ1-42 and phospho-Tau levels were increased in various PM exposure conditions compared to the clean air control. NanoString GeoMx analysis demonstrated a remarkable activation of immune responses in the PM exposed mouse brain. Surprisingly, our data also indicated a strong activation of various tumor suppressors including RB1, CDKN1A/p21 and CDKN2A/p16. Collectively, our data demonstrated that exposure to airborne PM caused a profound transcriptional dysregulation and accelerated Alzheimer's-related pathology.

Figure S10. Expression of Notch-1 and its ligand Jagged1 is significantly decreased in patient-derived glioblastoma cell lines with laminin-411 disruption by CRISPR/Cas9