Targeting Moiety Research Articles

B-Cell Receptor Antigens As the Targeting Moiety for Bispecific Constructs - a Novel Treatment Approach for B-Cell Lymphomas

BackgroundChronic antigenic stimulation of the B-cell receptor (BCR) has been suggested to play a role in the pathogenesis of B-cell lymphomas, however only few B-cell receptor antigens have been identified until recently, when we identified ARS2 and LRPAP1 as the autoantigenic targets of the B-cell receptors from approximately 25% of DLBCLs of the ABC type and 45% of mantle cell lymphomas, respectively. These BCR antigens can be used to therapeutically target lymphoma cells in an approach we designated as BARs (B-cell receptor antigens for reverse targeting). To test whether BARs can substitute for the B-cell targeting antibody CD19, we designed T-cell engaging anti-CD3 single chain fragments (scFv) and NK-cell engaging anti-CD16 scFv with the respective BAR as the targeting moiety.Material and methodsVariable heavy- and light- chain genes of the anti-CD3 OKT3 hybridoma and the anti-CD16 3G8 hybridoma were cloned into a pcDNA 3.1 vector by standard cloning techniques, followed by DNA sequences of the autoantigens ARS2 or LRPAP1. VH and VL were linked by a glycine-serine linker, as was VL to the autoantigenic epitope resulting in a VH-(Gly₄Ser₁)ⁿ-VL-(Gly₄Ser₁)ⁿ-ARS2/LRPAP1 DNA sequence. The final cloning product was transfected in HEK 293 cells for production of bispecific constructs. Binding capacity to lymphoma cell lines (DLBCL: OCI-Ly3, U2932, HBL-1; MCL : MAVER-1, GRANTA-519) and PBMCs was assessed by flow cytometry. Western blot analysis was used for detection of bispecific constructs after incubation with the monoclonal anti-His Tag antibody. Cytotoxicity was evaluated by LDH release assay.ResultsWe cloned, expressed, characterized and tested 4 bispecific constructs: αCD3-ARS2, αCD3-LRPAP1, αCD16-ARS2 and αCD16-LRPAP1. αCD3 bispecific constructs bound specifically to CD3 on T cells whereas αCD16 constructs bound exclusively to CD16-positive cells. Bispecific ARS2 constructs bound specifically to DLBCL cells with a B-cell receptor specific for ARS2, while LRPAP1 containing constructs bound exclusively to MCL with a B-cell receptor specific for LRPAP1. αCD3-ARS2 and αCD3-LRPAP1 induced dose-dependent T-cell mediated, rapid cytotoxicity exclusively in lymphoma cell lines with B-cell receptors specific for ARS2 and LRPAP1, respectively, starting at concentrations as low as 0,1 µg/ml. NK-cell mediated cytotoxicity against the respective DLBCL and MCL cell lines was observed with αCD16 constructs αCD16-ARS2 and αCD16-LRPAP1 at concentrations starting from 1 µg/ml. Control lymphoma cell lines with BCR specificities other than for ARS2 or LRPAP1 were not affected.ConclusionBARs can substitute for B-cell-directed antibodies (e. g. CD19) as the targeting moiety in bispecific constructs for the therapeutic targeting of B-cell lymphomas. Because approaches using the BCR antigen for targeting the malignant B cells have an exclusive specificity for the cells with the specific surface BCR (which are exclusively the respective lymphoma cells), they do not only represent a sensu stricto tumor-specific approach, they can also be expected to be more effective (due to the higher density of BCR molecules on the lymphoma cell surface), but less toxic than the currently available bispecific T-cell and NK-cell engaging antibodies, because they leave all other cells, in particular normal B-lymphocytes, unaffected. Supported by Wilhelm-Sander-Stiftung DisclosuresThurner:Saarland university: Patents & Royalties: Saarland university has applied for a relevant patent. Regitz:Saarland university: Patents & Royalties: Saarland university has applied for a relevant patent. Fadle:Saarland university: Patents & Royalties: Saarland university has applied for a relevant patent.

Tailorable Membrane-Penetrating Nanoplatform for Highly Efficient Organelle-Specific Localization.

Given the breadth of currently arising opportunities and concerns associated with nanoparticles for biomedical imaging, various types of nanoparticles have been widely exploited, especially for cellular/subcellular level probing. However, most currently reported nanoparticles either have inefficient delivery into cells or lack specificity for intracellular destinations. The absence of well-defined nanoplatforms remains a critical challenge hindering practical nano-based bio-imaging. Herein, the authors elaborate on a tailorable membrane-penetrating nanoplatform as a carrier with encapsulated actives and decorated surfaces to tackle the above-mentioned issues. The tunable contents in such a versatile nanoplatform offer huge flexibility to reach the expected properties and functions. Aggregation-induced emission luminogen (AIEgen) is applied to achieve sought-after photophysical properties, specific targeting moieties are installed to give high affinity towards different desired organelles, and critical grafting of cell-penetrating cyclic disulfides (CPCDs) to promote cellular uptake efficiency without sacrificing the specificity. Hereafter, to validate its practicability, the tailored nano products are successfully applied to track the dynamic correlation between mitochondria and lysosomes during autophagy. The authors believe that the strategy and described materials can facilitate the development of functional nanomaterials for various life science applications.

Enhancing Proteasome-Inhibitory Activity and Specificity of Bortezomib By Endocytosis-Mediated Uptake of CD38 Targeted Nanoparticles in Multiple Myeloma

The establishment of more effective treatments that can circumvent chemoresistance in Multiple Myeloma (MM) is a priority. Although bortezomib (BTZ) is one of the most potent proteasome inhibitors available, still possesses limitations related to dose limiting side effects. Several strategies have been developed to improve the delivery of chemotherapies to MM by targeting different moieties expressed on MM cells to nanoparticle delivery systems (NPs), which have failed mainly due to their heterogeneous expression on these cells. Our goal was to test CD38 targeted chitosan NPs as novel targeting moiety for MM to improve the potency and efficacy of BTZ in MM cells and reduce the side effects in healthy tissue. Due to its high expression on MM cells, CD38 is used as a marker for identification of MM cells, and there are several indications supporting the notion that CD38 plays significant roles in the progression of MM.We have fine-tuned the production of anti-CD38 chitosan NPs with a small size (50nm) to allow better penetration to tumors, and we showed that these NPs were stable at different storage conditions and periods. The chitosan NPs showed preferential drug release in MM tumor microenvironment compared to normal tissue microenvironment, and this release was pH dependent.The uptake of the anti-CD38 chitosan NPs to MM cells (cell lines and primary) was significantly higher than their uptake in normal cells (4-fold of BM MNCs, and 10-fold of PB MNCs) demonstrating that the anti-CD38 chitosan NPs are selective and specific to MM. BTZ-loaded anti-CD38 chitosan NPs demonstrated enhanced cytotoxicity and apoptosis on MM cells compared to similar concentration of BTZ as free drug; demonstrated by induction of more profound cell cycle arrest and apoptosis leading to cell death through inactivation of proliferative cell signaling pathways (MAPK, PI3K) and activation of pro-apoptotic pathways (PARP and Caspase-3). We also found that BTZ-loaded anti-CD38 NPs proved to be more efficacious than free BTZ and overcame CAM-DR and hypoxia-mediated drug resistance. The use of a 3DTEBM model, which recreate MM pathophysiology, corroborated the increase cytotoxicity of anti-CD38 chitosan NPs on MM cells relative to free drug and allowed to corroborated co-localization of the NPs in the MM cells.We found that BTZ-loaded NPs were significantly more potent in the proteasome activity inhibition than free BTZ and correlated with the boron content in MM cells treated with BTZ-loaded anti-CD38 NPs, which was 3-fold higher than in MM cells treated with BTZ as free drug. To confirm that the process involved in the internalization of the anti-CD38 chitosan NPs was endocytosis, we have shown that the NPs distribution in the cells co-localized with the distribution of endosomes and found that the anti-CD38 NPs were uptaken through clathrin-mediated and caveolae-mediated early endocytosis which progressed to late endocytosis and further delivery of the NPs content into the cells. Finally, we linked the internalization through endocytic pathways of anti-CD38 NPs to the enhanced proteasome activity inhibition of these NPs in MM cells. Treatment of MM cells with clathrin and caveolae-mediated early endocytosis, or late endocytosis inhibitors (but not macropinocytosis inhibitors) significantly reduced the proteasome inhibition of BTZ-loaded anti-CD38 chitosan NPs. When BTZ is administered as a free drug, it penetrated through passive diffusion into MM cells as well as in normal cells, and inhibition of the different endocytosis routes did not induce any change in its ability to inhibit the proteasome.In vivo studies showed that treatment with BTZ-loaded anti-CD38 targeted NPs (1mg/kg once a week) showed a markedly improved tumor growth inhibition, improved overall survival, and reduction of side effects compared to non-targeted NPs and free drug.In conclusion, anti-CD38 chitosan NPs showed preferential BTZ release in tumor-microenvironment, specific binding to MM cells, and an improved drug cellular uptake through endocytosis, which translated in enhanced proteasome inhibition and robust cytotoxic effect on MM cells. Furthermore, the anti-CD38 chitosan NPs specifically delivered therapeutic agents to MM cells improving therapeutic efficacy and reducing side effects in vivo. We are the first to report the enhancement of the efficacy and specificity of proteasome inhibitors due to endocytosis-driven delivery of NPs. DisclosuresDe La Puente:Cellatrix LLC: Other: Co-Founder. Vij:Celgene, Onyx, Takeda, Novartis, BMS, Sanofi, Janssen, Merck: Consultancy; Takeda, Onyx: Research Funding. Azab:Glycomimetics: Research Funding; Selexys: Research Funding; Karyopharm: Research Funding; Cellatrix LLC: Other: Founder and owner; Verastem: Research Funding; Cell Works: Research Funding; Abbvie: Research Funding; Tioma: Research Funding; Cleave Bioscience: Research Funding; Targeted Therapeutics LLC: Other: Founder and owner.

Effect of Protein Corona on Mitochondrial Targeting Ability and Cytotoxicity of Triphenylphosphonium Conjugated with Polyglycerol-Functionalized Nanodiamond.

Functionalization of nanoparticles (NPs) with targeting moieties has a high potential to advance precision nanomedicine. However, the targeting moieties on a NP surface are known to be masked by a protein corona in biofluids, lowering the targeting efficiency. Although it has been demonstrated at the cellular level, little is known about the influence of the protein corona on the subcellular targeting. Herein, we adopted triphenylphosphonium (TPP) as a mitochondrial targeting moiety and investigated the effects of protein coronas from fetal bovine serum and human plasma on its targeting ability and cytotoxicity. Specifically, we introduced TPP in low (l) and high (h) densities on the surface of nanodiamond (ND) functionalized with polyglycerol (PG). Despite the "corona-free" PG interface, we found that the TPP moiety attracted proteins to form a corona layer with clear linearity between the TPP density and the protein amount. By performing investigations on human cervix epithelium (HeLa) and human lung epithelial carcinoma (A549) cells, we further demonstrated that (1) the protein corona alleviated the cytotoxicity of both ND-PG-TPP-l and -h, (2) a smaller amount of proteins on the surface of ND-PG-TPP-l did not affect its mitochondrial targeting ability, and (3) a larger amount of proteins on the surface of ND-PG-TPP-h diminished its targeting specificity by restricting the NDs inside the endosome and lysosome compartments. Our findings will provide in-depth insights into the design of NPs with active targeting moiety for more precise and safer delivery at the subcellular level.

Mimicking Pathogens to Augment the Potency of Liposomal Cancer Vaccines.

Liposomes have emerged as interesting vehicles in cancer vaccination strategies as their composition enables the inclusion of both hydrophilic and hydrophobic antigens and adjuvants. In addition, liposomes can be decorated with targeting moieties to further resemble pathogenic particles that allow for better engagement with the immune system. However, so far liposomal cancer vaccines have not yet reached their full potential in the clinic. In this review, we summarize recent preclinical studies on liposomal cancer vaccines. We describe the basic ingredients for liposomal cancer vaccines, tumor antigens, and adjuvants, and how their combined inclusion together with targeting moieties potentially derived from pathogens can enhance vaccine immunogenicity. We discuss newly identified antigen-presenting cells in humans and mice that pose as promising targets for cancer vaccines. The lessons learned from these preclinical studies can be applied to enhance the efficacy of liposomal cancer vaccination in the clinic.

Lipoamino acid-modified GnRH analogs with receptor-mediated antiproliferative activity in prostate and ovarian cancer cells

Gonadotropin-releasing hormone (GnRH) analogs (e.g., triptorelin) are developed to treat hormone-dependent reproductive cancers. However, these analogs lack a significant direct antitumor activity to make them suitable for hormone-refractory reproductive cancers. In this study, we examined different biological properties of lipid-modified GnRH analogs, with/without D-amino acid substitution at position 6 in prostate and ovarian cancer cells. We revealed that the improved metabolic stability due to lipid-modification and D-amino acid substitution played a pivotal role in enhancing GnRH receptor-mediated direct antiproliferative activity up to 4.5-fold higher than triptorelin. Furthermore, a comparable FSH release and higher LH release activity in pituitary cells than triptorelin was observed, indicating an improved specificity and/or binding affinity to GnRH receptors. We confirmed the important role of sex steroids in the antitumor activity of the lipopeptides, which were contrasting in prostate and ovarian cancer cells. The superior activity of these GnRH analogs over commercial peptides renders promises for developing new GnRH receptor ligands to treat hormone-dependent and -refractory cancers, as well as emerging new targeting moieties for the delivery of anticancer agents in GnRH receptor-overexpressing cancers.

Development of mitochondrial targeted theranostic nanocarriers for treatment of gliomas

Theranostic nanoparticles were developed using synthesized Gadolinium doped iron oxide nanoplatforms by polyol process. Lenalidomide was coated on the magnetic core using PLGA by nanoprecipitation technique. Modification of Polyethylenimine was done with targeting moieties to provide targeting against gliomas. The cell line studies were performed on U87MG glioblastoma cell lines. Toxicity evaluation was done by mucosal toxicity study and hemolysis test. The magnetic core had ultra-small particle size of <10 nm and were paramagnetic with in vitro hyperthermia ability. The theranostic nanoparticles had high entrapment efficiency of 97.82 ± 0.425% and low particle size of 122.26 ± 2.17 nm. Cell line studies revealed the anti-cancer activity of formulation with localization in mitochondria. Toxicity studies on nasal mucosa and hemolysis test proved that the formulation was non-toxic as compared to positive control. Biodistribution studies on Wistar rats revealed the potential of the theranostic nanoparticles in brain targeting by intranasal route.

Nanomedicines functionalized with anti-EGFR ligands for active targeting in cancer therapy: Biological strategy, design and quality control

Recently, active targeting using nanocarriers with biological ligands has emerged as a novel strategy for improving the delivery of therapeutic and/or imaging agents to tumor cells. The presence of active targeting moieties on the surface of nanomedicines has been shown to play an important role in enhancing their accumulation in tumoral cells and tissues versus healthy ones. This property not only helps to increase the therapeutic index but also to minimize possible side effects of the designed nanocarriers. Since the overexpression of epidermal growth factor receptors (EGFR) is a common occurrence linked to the progression of a broad variety of cancers, the potential application of anti-EGFR immunotherapy and EGFR-targeting ligands in active targeting nanomedicines is getting increasing attention. Henceforth, the EGFR-targeted nanomedicines were extensively studied in vitro and in vivo but exhibited both satisfactory and disappointing results, depending on used protocols. This review is designed to give an overview of a variety of EGFR-targeting ligands available for nanomedicines, how to conjugate them onto the surface of nanoparticles, and the main analytical methods to confirm this successful conjugation.

Lysosome-targetable selenium-doped carbon nanodots for in situ scavenging free radicals in living cells and mice.

Lysosome-targetable selenium-doped carbon nanodots (Lyso-Se-CDs) that can efficiently scavenge lysosomal •OH in living cells and mice were designed in this research. Se-CDs with redox-responsive fluorescence (λex = 379 nm, λem = 471 nm, quantum yield = 7.1%) were initially synthesized from selenocystine by a facile hydrothermal method, followed by the surface modification with morpholine, a lysosome targeting moiety. The as-synthesized Lyso-Se-CDs exhibited excellent colloidal stability, efficient scavenging abilities towards •OH, low biotoxicity, as well as good biocompatibility and lysosome targetability. Due to these desirable properties, Lyso-Se-CDs had been successfully utilized for rescuing cells from elevated lysosomal •OH levels. More importantly, Lyso-Se-CDs efficiently relieved phorbol 12-myristate 13-acetate (PMA) triggered ear inflammation in live mice. These findings reveal that Lyso-Se-CDs are potent candidates for treating •OH-related inflammation.

Pharmacodynamic and Pharmacokinetic Properties of Full Phosphorothioate Small Interfering RNAs for Gene Silencing In Vivo

State-of-the-art small interfering RNA (siRNA) therapeutics such as givosiran and fitusiran are constructed from three variable components: a fully-modified RNA core that conveys metabolic stability, a targeting moiety that mediates target-cell uptake, and a linker. This structural complexity poses challenges for metabolite characterization and risk assessment after long-term patient exposure. In this study, we show that basic phosphorothioate modification of a siRNA targeting the oncoprotein Lin28B provides a useful increase in metabolic stability, without greatly compromising potency. We found that its stability in vitro matched that of nanoparticle-free patisiran in serum and surpassed it in liver tritosome extracts, although it did not reach the stability of the fitusiran siRNA core structure. Liver and kidney were the main sites of accumulation after its subcutaneous administration in mice. Despite the lack of a delivery agent-free antitumor effect, we anticipate our study to be a starting point to develop alternative siRNA scaffolds that can be degraded into naturally-occurring metabolites and help alleviate the aforementioned challenges. Furthermore, Lin28B is a promising target for cancers, and the development of such simplified siRNA analogs, possibly together with novel targeting units, holds potential.

Nanogold Functionalized With Lipoamide-isoDGR: A Simple, Robust and Versatile Nanosystem for αvβ3-Integrin Targeting.

Gold nanoparticles functionalized with isoDGR, a tripeptide motif that recognizes αvβ3 integrin overexpressed in tumor vessels, have been used as nano-vectors for the delivery of cytokines to tumors. Functionalization of nanogold with this peptide has been achieved by coating nanoparticles with a peptide-albumin conjugate consisting of heterogeneous molecules with a variable number of linkers and peptides. To reduce nanodrug heterogeneity we have designed, produced and preclinically evaluated a homogeneous and well-defined reagent for nanogold functionalization, consisting of a head-to-tail cyclized CGisoDGRG peptide (iso1) coupled via its thiol group to maleimide-PEG11-lipoamide (LPA). The resulting iso1-PEG11-LPA compound can react with nanogold via lipoamide to form a stable bond. In vitro studies have shown that iso1, after coupling to nanogold, maintains its capability to bind purified αvβ3 and αvβ3-expressing cells. Nanogold functionalized with this peptide can also be loaded with bioactive tumor necrosis factor-α (TNF) to form a bi-functional nanodrug that can be stored for three days at 37°C or >1 year at low temperatures with no loss αvβ3-binding properties and TNF-cytolytic activity. Nanoparticles functionalized with both iso1 and TNF induced tumor eradication in WEHI-164 fibrosarcoma-bearing mice more efficiently than nanoparticles lacking the iso1 targeting moiety. These results suggest that iso1-PEG11-LPA is an efficient and well-defined reagent that can be used to produce robust and more homogeneous nano-vectors for the delivery of TNF and other cytokines to αvβ3 positive cells.

The antigen-binding moiety in the driver's seat of CARs.

Immuno‐oncology has been at the forefront of cancer treatment in recent decades. In particular immune checkpoint and chimeric antigen receptor (CAR)‐T cell therapy have achieved spectacular results. Over the years, CAR‐T cell development has followed a steady evolutionary path, focusing on increasing T cell potency and sustainability, which has given rise to different CAR generations. However, there was less focus on the mode of interaction between the CAR‐T cell and the cancer cell; more specifically on the targeting moiety used in the CAR and its specific properties. Recently, the importance of optimizing this domain has been recognized and the possibilities have been exploited. Over the last 10 years—in addition to the classical scFv‐based CARs—single domain CARs, natural receptor‐ligand CARs, universal CARs and CARs targeting more than one antigen have emerged. In addition, the specific parameters of the targeting domain and their influence on T cell activation are being examined. In this review, we concisely present the history of CAR‐T cell therapy, and then expand on various developments in the CAR ectodomain. We discuss different formats, each with their own advantages and disadvantages, as well as the developments in affinity tuning, avidity effects, epitope location, and influence of the extracellular spacer.

Extracellular Vesicles as Potential Therapeutics for Inflammatory Diseases.

Extracellular vesicles (EV) deliver cargoes such as nucleic acids, proteins, and lipids between cells and serve as an intercellular communicator. As it is revealed that most of the functions associated to EVs are closely related to the immune response, the important role of EVs in inflammatory diseases is emerging. EVs can be functionalized through EV surface engineering and endow targeting moiety that allows for the target specificity for therapeutic applications in inflammatory diseases. Moreover, engineered EVs are considered as promising nanoparticles to develop personalized therapeutic carriers. In this review, we highlight the role of EVs in various inflammatory diseases, the application of EV as anti-inflammatory therapeutics, and the current state of the art in EV engineering techniques.

Current State of Breast Cancer Diagnosis, Treatment, and Theranostics.

Breast cancer is one of the leading causes of cancer-related morbidity and mortality in women worldwide. Early diagnosis and effective treatment of all types of cancers are crucial for a positive prognosis. Patients with small tumor sizes at the time of their diagnosis have a significantly higher survival rate and a significantly reduced probability of the cancer being fatal. Therefore, many novel technologies are being developed for early detection of primary tumors, as well as distant metastases and recurrent disease, for effective breast cancer management. Theranostics has emerged as a new paradigm for the simultaneous diagnosis, imaging, and treatment of cancers. It has the potential to provide timely and improved patient care via personalized therapy. In nanotheranostics, cell-specific targeting moieties, imaging agents, and therapeutic agents can be embedded within a single formulation for effective treatment. In this review, we will highlight the different diagnosis techniques and treatment strategies for breast cancer management and explore recent advances in breast cancer theranostics. Our main focus will be to summarize recent trends and technologies in breast cancer diagnosis and treatment as reported in recent research papers and patents and discuss future perspectives for effective breast cancer therapy.

Nanoparticles: Untying the Gordian Knot in Conventional Computed Tomography Imaging

X-ray computed tomography (CT) imaging plays an essential role in disease diagnosis due to its noninvasive, painless mode and superior penetration depth. However, the resolution of the soft tissue ...

Novel decorated nanostructured lipid carrier for simultaneous active targeting of three anti-cancer agents

Cancer-targeted co-delivery of therapeutic agents has been recognized as an effective strategy for increasing efficacy and reducing side effects of therapeutic agents. In this study, we used methotrexate (MTX) alone as a targeting moiety and chemotherapeutic agent and in combination with docetaxel (DTX) and doxorubicin (DOX) as chemotherapeutic agents to stop cancer cell proliferation with the aid of newly designed nanostructured lipid carriers (NLCs). The physicochemical properties of our designed nanocomplexes were evaluated by DLS, FT-IR spectroscopy, SEM, and TEM. Moreover, the targeting efficiency of the designed and synthesized nanoplatforms was evaluated on the folate receptor (FR) positive human breast cancer cell line (MCF-7) and FR negative human alveolar basal epithelial cells (A549). The NLCs/DTX/DOX/CS and NLCs/DTX/DOX/CS-MTX complexes significantly increased the cell cytotoxicity and the cell apoptosis rate. However, the complexes significantly reduced the capability of colony formation and cell migration. Our results revealed that NLCs/DTX/DOX/CS-MTX had synergistic cytotoxicity, reactive oxygen spaces, autophagy, and the apoptosis induction ability with an enhanced cellular internalization rate in FR-positive cancer cells, thorough MTX recognition capability. We conclude that the NLCs/DTX/DOX/CS-MTX complex is a new promising paradigm for breast cancer-targeted co-delivery.

Traceless Delivery of 4‐Hydroxytamoxifen for Controllable Gene Expression in Astrocytes

Astrocytes are the most abundant type of non-neuronal cells in the brain. In addition to maintaining blood-brain barrier integrity and providing nutrients to the surrounding cells, they play an active role in brain function by the uptake and release of neurotransmitters through the tripartite synapse. Astrocytes play a crucial role in the maintenance of the health and function of the central nervous system and thus astrocytic dysfunction has been implicated in many neurological diseases like Parkinson's disease (PD), Alzheimer's disease (AD), Amyotrophic lateral sclerosis (ALS), glioblastoma and depression. Hence, the delivery of small molecule drugs specifically to astrocytes in coordination with gene therapy is a potential therapeutic tool to activate the expression of therapeutic genes to re-establish healthy astrocytic functions. Recently, a targeting moiety was developed in our group that transports small molecule cargo to astrocytes. Here we adapt the astrocyte targeting moiety to deliver the drug tamoxifen, which is widely used for controlling gene expression in rodents. Since tamoxifen does not have a potential site of modification to attach the tag while retaining the activity of the molecule, we explored a traceless delivery strategy. The active metabolite 4-hydroxytamoxifen will be caged with a linker attached to the targeting moiety, where the release of the active drug will occur in the presence of light when delivered to astrocytes. The traceless targeted delivery strategy will help us to evaluate the ability of the targeting moiety to deliver drugs to astrocytes and to explore the release of different drug candidates in the native form inside astrocytes. Additionally, it will open the door to study the controlled gene expression of specific genes in astrocytes using the tamoxifen inducible Cre-ER/loxP system. In the future, the molecules we describe have potential use in gene therapy applications for exploring therapeutic aspects of astrocytes in various neurological diseases.

Development of Relaxin‐Conjugated Nanoparticles to Target and Activate RXFP1 in Hepatic Stellate Cells

Background Hepatic fibrosis is a progressive disease which can result in end-stage cirrhosis, a leading cause of death in the US. It is caused by the activation of myofibroblasts (HSC), that produce excess collagen under perpetual injury. Relaxin, a member of the insulin/IGF/relaxin superfamily of peptide hormones, has been shown to play a role in deactivating HSC through its receptor, RXFP1. The deactivation of HSC reduces expression of collagen, therefore slowing the progression of fibrosis. Significance of Problem Relaxin has been shown to be antifibrotic in some rodent models of hepatic fibrosis, however it is unstable in circulation and requires long-term continuous treatment. By developing a stabilized transport system, it would improve the efficacy and provide an HSC-specific targeting system through RXFP1. Hypothesis Relaxin tethered to nanoparticles (NP) will direct the particles to the HSC and activate RXFP1, thereby inducing an antifibrotic response. Experimental Design NP were prepared from rhodamine-labled poly(lactide-co-glycolide) (PLGA 16kDa)-b-PEG (3.4kDa)-COOH copolymers to produce control NP (C-NP). Relaxin was attached to the carboxylic group by carbodiimide crosslinking (R-NP). The particles were subjected to hydrodynamic analysis to determine their size. Human HEK-293T cells stably expressing RXFP1 (HEK-RXFP1) were used for NP binding. RXFP1 activation in HEK-RXFP1 cells, control cells (HEK), and the hepatic stellate cell line, LX2, which endogenously expresses RXFP1, was determined by cAMP production using a TR-FRET assay. To determine the localization of NP in diseased liver, hepatic fibrosis was induced in mice by injection of carbon tetrachloride for 2 weeks. NP were injected via the tail vein, and 2 hours later the liver and other organs were removed, and fluorescence detected using an IVIS imager. Frozen sections were used to determine colocalization of NP with cell marker proteins by fluorescence immunohistochemistry. Results/Data The conjugation of relaxin caused a small increase in particle size. TR-FRET analysis showed R-NP increase cAMP production, indicative of RXFP1 activation. HEK cells showed no response to either NP, however HEK-RXFP1 cells produced a concentration-dependent response to R-NP. Similarly, R-NP induced a dose-dependent cAMP increase in LX2 cells. Contrary to the C-NP, there was increased binding of R-NP to HEK-RXFP1 cells. IVIS imaging showed that both NP homed primarily to the liver, however R-NP had increased homing to kidney and adipose tissue, other sites of RXFP1 expression. Immunohistochemistry revealed that C-NP primarily colocalized with macrophages in the liver, while R-NP had increased association with HSC. Conclusions This study showed that covalent attachment of relaxin to NP retains biological activity and allows for HSC targeting both in vitro and in vivo. This proof-of-concept shows that R-NP can be used as both a targeting moiety and antifibrotic agent. Future studies aim to optimize the configurations of the NP and load antifibrotic agents as cargo to be delivered to desired profibrotic cells.

Ru(II) Polypyridine Complex-Functionalized Mesoporous Silica Nanoparticles as Photosensitizers for Cancer Targeted Photodynamic Therapy

Cancer is the leading cause of death in the developed world. In the last few decades, photodynamic therapy (PDT) has augmented the number of medical techniques to treat this disease in the clinics. As the pharmacological active species to kill cancer cells are only generated upon light irradiation, PDT is associated with an intrinsic first level of selectivity. However, since PDT agents also accumulate in the surrounding, healthy tissue and since it is practically very challenging to only expose the tumor site to light, some side effects can be observed. Consequently, there is a need for a selective drug delivery system, which would give a second level of selectivity. In this work, a dual tumor targeting approach is presented based on mesoporous silica nanoparticles, which act by the enhanced permeability and retention effect, and the conjugation to folic acid, which acts as a targeting moiety for folate receptor-overexpressed cancer cells. The conjugates were found to be nontoxic in noncancerous human normal lung fibroblast cells while showing a phototoxic effect upon irradiation at 480 or 540 nm in the low nanomolar range in folate receptor overexpressing cancerous human ovarian carcinoma cells, demonstrating their potential for cancer targeted treatment.

Imaging in vivo acetylcholine release in the peripheral nervous system with a fluorescent nanosensor

The ability to monitor the release of neurotransmitters during synaptic transmission would significantly impact the diagnosis and treatment of neurological diseases. Here, we present a DNA-based enzymatic nanosensor for quantitative detection of acetylcholine (ACh) in the peripheral nervous system of living mice. ACh nanosensors consist of DNA as a scaffold, acetylcholinesterase as a recognition component, pH-sensitive fluorophores as signal generators, and α-bungarotoxin as a targeting moiety. We demonstrate the utility of the nanosensors in the submandibular ganglia of living mice to sensitively detect ACh ranging from 0.228 to 358 μM. In addition, the sensor response upon electrical stimulation of the efferent nerve is dose dependent, reversible, and we observe a reduction of ∼76% in sensor signal upon pharmacological inhibition of ACh release. Equipped with an advanced imaging processing tool, we further spatially resolve ACh signal propagation on the tissue level. Our platform enables sensitive measurement and mapping of ACh transmission in the peripheral nervous system.