Aptamer-Assisted Traceless Isolation of PD-L1-Positive Small Extracellular Vesicles for Dissecting Their Subpopulation Signature and Function.

Small extracellular vesicles (sEVs) are heterogeneous membrane-bound vesicles that carry numerous bioactive molecules. Studies have reported that sEVs carrying PD-L1 on the surface could contribute to immunosuppression; however, the precise mechanisms are unclear. To fully dissect their mode of action, it requires qualified methods to specifically isolate natural PD-L1-positive sEVs from heterogeneous sEVs. This study reported an aptamer-assisted capture-and-release strategy for traceless isolation of PD-L1-positive sEVs. The PD-L1 aptamer-anchored magnetic microspheres enable the specific capture of PD-L1-positive sEVs. The traceless release of captured PD-L1-positive sEVs was triggered by competition of complementary oligonucleotides, endowing the obtained label-free PD-L1-positive sEVs with natural properties. Benefited from this traceless isolation strategy, the distinct molecule profiles in adhesion and immuno-regulation between PD-L1-positive and PD-L1-negative sEVs were revealed. Compared to PD-L1-negative sEVs, PD-L1-positive sEVs were much more concentrated in cadherin binding, accompanied by increased adhesion to lymphatic endothelial cells and T cells but decreased adhesion to the extracellular matrix. Moreover, PD-L1-positive sEVs could transfer their enriched immunosuppressive "synapse"-related proteins to antigen-presenting cells, thereby inducing a tolerogenic-like phenotype. In summary, the present work dissects the subpopulation signature and action mode of PD-L1-positive sEVs for the first time and provides a general approach to the traceless isolation of sEV subpopulations.

Antifungal activity of montmorillonite/peptide aptamer nanocomposite against Colletotrichum gloeosporioides on Stylosanthes.

Chemical agents are effective treatment methods for anthracnose induced by pathogenic Colletotrichum gloeosporioides on Stylosanthes. However, excess consumption of chemical agents destroys the environment, synthetic biology was capable of conquering the issue. The antifungal agent is developed by enclosing a bio-synthesized peptide aptamer with layered montmorillonite via electrostatic interaction. Compared with free peptide aptamer, the nanocomposite exhibits higher antifungal activity against Colletotrichum gloeosporioides, further improving the utilization of peptide aptamer. The nanocomposite killed Colletotrichum gloeosporioides by releasing peptide aptamer after they entered the spore. Moreover, montmorillonite enhances the adhesion ability of peptide aptamer via hydrophobic interactions between nanomaterials and leaves, prolonging the extension time of nanocomposite on leaves. Consequently, 0.1 mg of nanocomposite demonstrates a comparable effect to commercial carbendazim (1 %) to prevent anthracnose on leaves of Stylosanthes induced by HK-04 at room temperature. This work demonstrates an alternative to commercial antifungal agents and proposes a versatile approach to preparing environmental-friendly antifungal agents to inhibit fungal infections on crops.

Targeting intrinsically disordered regions facilitates discovery of calcium channels 3.2 inhibitory peptides for adeno-associated virus-mediated peripheral analgesia.

ABSTRACT: Ample data support a prominent role of peripheral T-type calcium channels 3.2 (Ca V 3.2) in generating pain states. Development of primary sensory neuron-specific inhibitors of Ca V 3.2 channels is an opportunity for achieving effective analgesic therapeutics, but success has been elusive. Small peptides, especially those derived from natural proteins as inhibitory peptide aptamers (iPAs), can produce highly effective and selective blockade of specific nociceptive molecular pathways to reduce pain with minimal off-target effects. In this study, we report the engineering of the potent and selective iPAs of Ca V 3.2 from the intrinsically disordered regions (IDRs) of Ca V 3.2 intracellular segments. Using established prediction algorithms, we localized the IDRs in Ca V 3.2 protein and identified several Ca V 3.2iPA candidates that significantly reduced Ca V 3.2 current in HEK293 cells stably expressing human wide-type Ca V 3.2. Two prototype Ca V 3.2iPAs (iPA1 and iPA2) derived from the IDRs of Ca V 3.2 intracellular loops 2 and 3, respectively, were expressed selectively in the primary sensory neurons of dorsal root ganglia in vivo using recombinant adeno-associated virus (AAV), which produced sustained inhibition of calcium current conducted by Ca V 3.2/T-type channels and significantly attenuated both evoked and spontaneous pain behavior in rats with neuropathic pain after tibial nerve injury. Recordings from dissociated sensory neurons showed that AAV-mediated Ca V 3.2iPA expression suppressed neuronal excitability, suggesting that Ca V 3.2iPA treatment attenuated pain by reversal of injury-induced neuronal hypersensitivity. Collectively, our results indicate that Ca V 3.2iPAs are promising analgesic leads that, combined with AAV-mediated delivery in anatomically targeted sensory ganglia, have the potential to be a selective peripheral Ca V 3.2-targeting strategy for clinical treatment of pain.

Antifungal activity of an artificial peptide aptamer SNP-D4 against Fusarium oxysporum.

Fusarium oxysporum f. sp. cubense (FOC4) is a pathogen of banana fusarium wilt, which is a serious problem that has plagued the tropical banana industry for many years. The pathogenic mechanism is complex and unclear, so the prevention and control in agricultural production applications is ineffective. SNP-D4, an artificial peptide aptamer, was identified and specifically inhibited FOC4. To evaluate the efficacy of SNP-D4, FoC4 spores were treated with purified SNP-D4 to calculate the germination and fungicide rates. Damage of FOC4 spores was observed by staining with propidium iodide (PI). Eight proteins of FOC4 were identified to have high affinity for SNP-D4 by a pull-down method combined with Q-Exactive mass spectrometry. Of these eight proteins, A0A5C6SPC6, the aldehyde dehydrogenase of FOC4, was selected as an example to scrutinize the interaction sites with SNP-D4. Molecular docking revealed that Thr66 on the peptide loop of SNP-D4 bound with Tyr437 near the catalytic center of A0A5C6SPC6. Subsequently 42 spore proteins which exhibited associations with the eight proteins were retrieved for protein-protein interaction analysis, demonstrating that SNP-D4 interfered with pathways including 'translation', 'folding, sorting and degradation', 'transcription', 'signal transduction' and 'cell growth and death', eventually causing the inhibition of growth of FOC4. This study not only investigated the possible pathogenic mechanism of FOC4, but also provided a potential antifungal agent SNP-D4 for use in the control of banana wilt disease.

The aptamer BT200 blocks von Willebrand factor and platelet function in blood of stroke patients.

The effect of conventional anti-platelet agents is limited in secondary stroke prevention, and their effects are blunted under high shear stress in the presence of increased levels of circulating von Willebrand factor (VWF). VWF is critically involved in thrombus formation at sites of stenotic extracranial/intracranial arteries. A third generation anti-VWF aptamer (BT200) has been generated which could be useful for secondary stroke prevention. To characterize the effects of BT200 in blood of patients with large artery atherosclerosis stroke (LAA). Blood samples were obtained from 33 patients with acute stroke or transient ischemic attack to measure inhibition of VWF activity and VWF-dependent platelet function. Patients who received clopidogrel or dual antiplatelet therapy did not differ in VWF dependent platelet function tests from aspirin treated patients. Of 18 patients receiving clopidogrel with or without aspirin, only 3 had a prolonged collagen adenosine diphosphate closure time, and none of the patients had ristocetin induced aggregation in the target range. BT200 concentration-dependently reduced median VWF activity from 178 to < 3%, ristocetin induced platelet aggregation from 40U to < 10U and prolonged collagen adenosine diphosphate closure times from 93 s to > 300 s. Baseline VWF activity correlated (r = 0.86, p < 0.001) with concentrations needed to reduce VWF activity to < 20% of normal, indicating that BT200 acts in a target concentration-dependent manner. Together with a long half-life supporting once weekly administration, the safety and tolerability observed in an ongoing phase I trial, and the existence of a reversal agent, BT200 is an interesting drug candidate.

Role of the C5a-C5a receptor axis in the inflammatory responses of the lungs after experimental polytrauma and hemorrhagic shock.

Singular blockade of C5a in experimental models of sepsis is known to confer protection by rescuing lethality and decreasing pro-inflammatory responses. However, the role of inhibiting C5a has not been evaluated in the context of sterile systemic inflammatory responses, like polytrauma and hemorrhagic shock (PT + HS). In our presented study, a novel and highly specific C5a L-aptamer, NoxD21, was used to block C5a activity in an experimental murine model of PT + HS. The aim of the study was to assess early modulation of inflammatory responses and lung damage 4 h after PT + HS induction. NoxD21-treated PT + HS mice displayed greater polymorphonuclear cell recruitment in the lung, increased pro-inflammatory cytokine levels in the bronchoalveolar lavage fluids (BALF) and reduced myeloperoxidase levels within the lung tissue. An in vitro model of the alveolar-capillary barrier was established to confirm these in vivo observations. Treatment with a polytrauma cocktail induced barrier damage only after 16 h, and NoxD21 treatment in vitro did not rescue this effect. Furthermore, to test the exact role of both the cognate receptors of C5a (C5aR1 and C5aR2), experimental PT + HS was induced in C5aR1 knockout (C5aR1 KO) and C5aR2 KO mice. Following 4 h of PT + HS, C5aR2 KO mice had significantly reduced IL-6 and IL-17 levels in the BALF without significant lung damage, and both, C5aR1 KO and C5aR2 KO PT + HS animals displayed reduced MPO levels within the lungs. In conclusion, the C5aR2 could be a putative driver of early local inflammatory responses in the lung after PT + HS.

NoPv1: a synthetic antimicrobial peptide aptamer targeting the causal agents of grapevine downy mildew and potato late blight.

Grapevine (Vitis vinifera L.) is a crop of major economic importance. However, grapevine yield is guaranteed by the massive use of pesticides to counteract pathogen infections. Under temperate-humid climate conditions, downy mildew is a primary threat for viticulture. Downy mildew is caused by the biotrophic oomycete Plasmopara viticola Berl. & de Toni, which can attack grapevine green tissues. In lack of treatments and with favourable weather conditions, downy mildew can devastate up to 75% of grape cultivation in one season and weaken newly born shoots, causing serious economic losses. Nevertheless, the repeated and massive use of some fungicides can lead to environmental pollution, negative impact on non-targeted organisms, development of resistance, residual toxicity and can foster human health concerns. In this manuscript, we provide an innovative approach to obtain specific pathogen protection for plants. By using the yeast two-hybrid approach and the P. viticola cellulose synthase 2 (PvCesA2), as target enzyme, we screened a combinatorial 8 amino acid peptide library with the aim to identify interacting peptides, potentially able to inhibit PvCesa2. Here, we demonstrate that the NoPv1 peptide aptamer prevents P. viticola germ tube formation and grapevine leaf infection without affecting the growth of non-target organisms and without being toxic for human cells. Furthermore, NoPv1 is also able to counteract Phytophthora infestans growth, the causal agent of late blight in potato and tomato, possibly as a consequence of the high amino acid sequence similarity between P. viticola and P. infestans cellulose synthase enzymes.

A PD-L1 Aptamer Selected by Loss-Gain Cell-SELEX Conjugated with Paclitaxel for Treating Triple-Negative Breast Cancer.

BACKGROUND The clinical challenges of triple-negative breast cancer (TNBC) includes the lack of targeted therapy and chemoresistance. TNBC has relatively high PD-L1 expression, and PD-L1 antibody in combination with nab-paclitaxel has been approved by FDA for TNBC treatment. Aptamers, also termed chemical antibody, are widely used in targeted drug delivery. The present study aimed to select a DNA aptamer that could specifically bind and deliver drugs to TNBC cells. MATERIAL AND METHODS An innovative loss-gain cell-SELEX strategy was used to select DNA aptamer for PD-L1 protein. Construction of PD-L1 knock-out and over-expression MDA-MB-231 cell lines were conducted through transfection and confirmed by western blot and flow cytometry. Confocal microscopy and flow cytometry were used to analyze the binding ability of aptamer with TNBC cells. The cytotoxicity of aptamer-paclitaxel complex against TNBC cells was evaluated by Cell Counting Kit-8 assay. The reactivation of the T cell function by aptamer was measured by IL-2 enzyme-linked immunosorbent assay after T cells co-cultured with tumor cells. RESULTS In this work, using an innovative loss-gain cell-SELEX strategy, we screened a PD-L1-targeting aptamer. PD-L1 aptamer selectively bound to PD-L1 over-expressed TNBC cells with a dissociation constant in the nanomolar range. PD-L1 aptamer could also inhibit PD-1/PD-L1 interaction and restore the function of T cells. Moreover, we developed a PD-L1 aptamer-paclitaxel conjugate which showed improved cellular uptake and anti-proliferation efficacy in PD-L1 over-expressed TNBC cells. CONCLUSIONS In summary, these findings suggest that the selected PD-L1 aptamer might have potential implication in immune modulation and targeted therapy against TNBC.

Targeting SOX2 Protein with Peptide Aptamers for Therapeutic Gains against Esophageal Squamous Cell Carcinoma.

Esophageal squamous cell carcinoma (ESCC) is a predominant cancer type in developing countries such as China, where ESCC accounts for approximately 90% of esophageal malignancies. Lacking effective and targeted therapy contributes to the poor 5-year survival rate. Recent studies showed that about 30% of ESCC cases have high levels of SOX2. Herein, we aim to target this transcription factor with aptamer. We established a peptide aptamer library and then performed an unbiased screening to identify several peptide aptamers including P42 that can bind and inhibit SOX2 downstream target genes. We further found that P42 overexpression or incubation with a synthetic peptide 42 inhibited the proliferation, migration, and invasion of ESCC cells. Moreover, peptide 42 treatment inhibited the growth and metastasis of ESCC xenografts in mouse and zebrafish. Further analysis revealed that P42 overexpression led to alternations in the levels of proteins that are important for the proliferation and migration of ESCC cells. Taken together, our study identified the peptide 42 as a key inhibitor of SOX2 function, reducing the proliferation and migration of ESCC cells in vitro and in vivo, and thereby offering a potential therapy against ESCC.

Anti-VEGF-Aptamer Modified C-Dots-A Hybrid Nanocomposite for Topical Treatment of Ocular Vascular Disorders.

The vascular endothelial growth factor (VEGF) induces pathological angiogenetic ocular diseases. It is a scientific challenge to develop carriers for the controlled release of inhibitors for VEGF present in the back of the eye domain. Carbon dots (C-dots) functionalized with the VEGF aptamer are introduced and the hybrid nanoparticles are used for ocular nanomedicine. The C-dots are applied as effective carriers of the anti-VEGF aptamer across the cornea, yielding therapeutic levels upon topical administration. The hybrids show no toxicity for both in vitro and in vivo murine animal model, and further enable noninvasive intraocular concentration monitoring through the C-dots inherent fluorescence. In addition, the hybrid C-dots effectively inhibit VEGF-stimulated angiogenesis in choroidal blood vessels. This inhibition is comparable to two commercially available anti-VEGF drugs, bevacizumab and aflibercept. The hybrid aptamer-modified C-dots provide a versatile nanomaterial to treat age-related macular degeneration and diabetic retinopathy.

SS30, a novel thioaptamer targeting CD123, inhibits the growth of acute myeloid leukemia cells.

AIMS: CD123 represents an important acute myeloid leukemia (AML) therapeutic target. CD123 aptamers may potentially serve as tumor-homing ligands with excellent affinity and specificity for AML targeted therapy, but their complexity, laborious preparation and nuclease digestion limited pharmacological application. The aim of this study was to develop the first CD123 thioaptamer to overcome these obstacles. MAIN METHODS: Flow cytometry was utilized to assess the binding specificity, affinity and anti-nuclease ability of thioaptamer. CCK8, Annexin-V/DAPI, and colony forming assays were used to evaluate the anti-cancer ability of thioaptamer in vitro. The tumor volume, weights, survival rate, H&E staining of organs, and serum level of organ damage biomarkers of animal model were applied to investigate the anti-cancer ability of thioaptamer in vivo. Furthermore, we explored the binding mechanism between thioaptamer and CD123. KEY FINDINGS: CD123 thioaptamer SS30 was able to bind to CD123 structure with high specificity in complex nuclease environment, the dissociation constant of 39.1 nM for CD123 peptide and 287.6 nM for CD123+ AML cells, while exhibiting minimal cross-reactivity to albumin. Furthermore, SS30 inhibited the proliferation and survival of AML cell lines and human AML blasts selectively in vitro (P < 0.01). In addition, SS30 prolonged the survival and inhibited tumor growth in a mouse xenograft tumor model in vivo. Of note, SS30 blocked the interaction between IL-3 and CD123, and decreased expression of p-STAT5 and p-AKT. SIGNIFICANCE: The proliferation inhibition and nuclease resistance ability of SS30 made it as a more promising functional molecule for AML targeted therapy.

Engineering a peptide aptamer to target calmodulin for the inhibition of Magnaporthe oryzae.

To develop an antimicrobial agent for preventing the devasting damage caused by rice blast, a novel peptide aptamer was identified to interact with calmodulin (CaM) for the inhibition of the spore development in the pathogen Magnaporthe oryzae. A peptide aptamer designated as SNP-D4, consisted of the scaffold protein Staphylococcus aureus nuclease (SN) and an exposed surface loop of 16 random amino acids, was screened from the constructed peptide aptamer libraries by bacterial two-hybrid system using CaM of M. oryzae as the bait. The preliminary inhibition in the sporulation development was observed after treating with the crude extracts expressing SNP-D4. The inhibition efficacies of the purified SNP-D4 were quantified at the stages of conidial germination, germ tube elongation, and appressorium formation in M. oryzae. The binding affinity analysis revealed that SNP-D4 interacted with CaM at a dissociation constant (Kd) of about 20 µM. Moreover, the N-terminus of CaM was identified as the key binding region.

Studies on effectiveness of PTT on 3D tumor model under microfluidic conditions using aptamer-modified nanoshells.

Herein, we present the research focused on the synthesis and application of aptamer-modified gold nanoshells for photothermal therapy (PTT). NIR-absorbing hollow gold nanoshells were synthetized and conjugated with anti-MUC1 aptamer (HGNs@anti-MUC1). MUC1 (Mucin 1) is a transmembrane glycoprotein, which is overexpressed in a variety of epithelial cancers (eg. breast, lung, pancreatic). In order to evaluate the efficiency of PTT with HGNs@anti-MUC1 we used 3D cell culture model - multicellular spheroids. The selected cell culture model is considered as the best in vitro model for cancer research (similar morphology, metabolite and oxygen gradients, cellular interactions and cell growth kinetics in the spheroids are similar to the early stage of a nonvascular tumor). We conducted our research on human normal (MRC-5, MCF-10A) and tumor (A549, MCF-7) cell lines using a microfluidic system. Aptamer-modified nanoparticles were accumulated selectively in tumor cells (A549, MCF-7) and this fact contributed to the reduction of tumor spheroids viability and size. It should be underlined, that it is the first example of photothermal therapy carried out in a microsystem on multicellular spheroids.

Cascaded Aptamers-Governed Multistage Drug-Delivery System Based on Biodegradable Envelope-Type Nanovehicle for Targeted Therapy of HER2-Overexpressing Breast Cancer.

Tumor-specific therapeutic platforms with improved targeting efficacy and minimized side effect are crucial in cancer therapy. Capitalizing on the recognition capability and biocompatibility of aptamers, we herein designed a multistage targeted drug-delivery system using multiple biodegradable molecules-enveloped nanovehicle that can be employed to efficiently treat human epithelial growth factor receptor (HER2)-overexpressing breast cancer. In this nanovehicle, two aptamers respectively specific to HER2 and ATP were organized in a hierarchical manner. The outmost HER2 aptamer (HB5) governs the recognition to HER2 protein overexpressed in SK-BR-3 cell lines, while the ATP aptamer incorporated with anticancer drug (-)-epigallocatechin gallate (EGCG) and protamine sulfate in the inner core functions as a switch of drug release in response to abundant intracellular ATP. The targeting and drug locker aptamers were cascaded for active targeting effect and stimuli responsiveness, guaranteeing the site-specific drug transportation and endogenous species-triggered drug release inside the tumor cells. Moreover, nanostructured lipid carriers (NLCs) were constructed to wrap and stabilize the loosely bounded ternary complex, minimizing premature drug leakage potentially encountered by the biomolecule assembled nanocarriers. This multiple biomolecules-enveloped nanovehicle demonstrated improved inhibitory actions on tumor growth and minimum side effect to normal organs and tissues both in vitro and in vivo. The presented nanovehicle built from recognition and therapeutic components in a nontoxic framework offered a promising drug-delivery platform with transport precision and biological safety.

Aptamer-Based Biosensors for Antibiotic Detection: A Review.

Antibiotic resistance and, accordingly, their pollution because of uncontrolled usage has emerged as a serious problem in recent years. Hence, there is an increased demand to develop robust, easy, and sensitive methods for rapid evaluation of antibiotics and their residues. Among different analytical methods, the aptamer-based biosensors (aptasensors) have attracted considerable attention because of good selectivity, specificity, and sensitivity. This review gives an overview about recently-developed aptasensors for antibiotic detection. The use of various aptamer assays to determine different groups of antibiotics, like ß-lactams, aminoglycosides, anthracyclines, chloramphenicol, (fluoro)quinolones, lincosamide, tetracyclines, and sulfonamides are presented in this paper.

Regulation of C-C chemokine receptor 5 (CCR5) stability by Lys197 and by transmembrane protein aptamers that target it for lysosomal degradation.

C-C motif chemokine receptor 5 (CCR5) is a cell surface-associated, immune-regulatory G protein-coupled receptor (GCPR) with seven transmembrane helices. We previously reported the isolation and initial characterization of short artificial transmembrane protein aptamers, named "traptamers," that specifically down-regulate CCR5 expression and inhibit infection of human T cells by HIV strains that use CCR5 as a co-receptor. Here, we investigated the mechanism of traptamer-mediated CCR5 down-regulation and show that most of the traptamers (designated class 1 traptamers) form a stable complex with CCR5 and target it for lysosome-mediated degradation. The ability of these traptamers to down-regulate CCR5 depended on Lys197 in the fifth transmembrane helix of CCR5. In the absence of traptamers, substitution of Lys197 to an uncharged amino acid increased CCR5 stability, and introduction of a lysine at the homologous position in CCR2b, a related chemokine receptor, decreased CCR2b levels. The prototypic class 2 traptamer BY6M4 also formed a complex with CCR5, but CCR5 down-regulation caused by class 2 traptamers did not depend on the lysosome or on Lys197 These results demonstrate that traptamers use diverse mechanisms to down-regulate CCR5 and identify a specific amino acid that plays a central role in controlling chemokine receptor stability. Further studies of these traptamers are likely to provide new insights into CCR5 metabolism and biology and may suggest new therapeutic approaches to modulate the levels of CCR5 and other GPCRs.

RAGE-aptamer attenuates deoxycorticosterone acetate/salt-induced renal injury in mice.

The mineralocorticoid receptor (MR) and its downstream signaling play an important role in hypertensive renal injury. The interaction of advanced glycation end products (AGE) with their receptor (RAGE) is involved in the progression of renal disease. However, the pathological crosstalk between AGE-RAGE axis and MR system in kidney derangement remains unclear. We screened DNA-aptamer directed against RAGE (RAGE-apt) in vitro and examined its effects on renal injury in uninephrectomized deoxycorticosterone acetate (DOCA)/salt-induced hypertensive mice. RAGE, GTP-bound Rac-1 (Rac1), and MR were co-localized in the podocytes of DOCA mice. The deletion of RAGE gene significantly inhibited mesangial matrix expansion and tubulointerstitial fibrosis in DOCA mice, which was associated with the reduction of glomerular oxidative stress, MR, Rac1, and urinary albumin excretion (UAE) levels. RAGE-apt attenuated the increase in carboxymethyllysine (CML), RAGE, nitrotyrosine, Rac1, and MR levels in the kidneys and reduced UAE in DOCA mice. Aldosterone (Aldo) increased nitrotyrosine, CML, and RAGE gene expression in murine podocytes, whereas CML stimulated MR and Rac1 levels, which were blocked by RAGE-apt. The present study indicates the crosstalk between the AGE-RAGE axis and Aldo-MR system, suggesting that RAGE-apt may be a novel therapeutic tool for the treatment of MR-associated renal diseases.

A water-soluble nucleolin aptamer-paclitaxel conjugate for tumor-specific targeting in ovarian cancer.

Paclitaxel (PTX) is among the most commonly used first-line drugs for cancer chemotherapy. However, its poor water solubility and indiscriminate distribution in normal tissues remain clinical challenges. Here we design and synthesize a highly water-soluble nucleolin aptamer-paclitaxel conjugate (NucA-PTX) that selectively delivers PTX to the tumor site. By connecting a tumor-targeting nucleolin aptamer (NucA) to the active hydroxyl group at 2' position of PTX via a cathepsin B sensitive dipeptide bond, NucA-PTX remains stable and inactive in the circulation. NucA facilitates the uptake of the conjugated PTX specifically in tumor cells. Once inside cells, the dipeptide bond linker of NucA-PTX is cleaved by cathepsin B and then the conjugated PTX is released for action. The NucA modification assists the selective accumulation of the conjugated PTX in ovarian tumor tissue rather than normal tissues, and subsequently resulting in notably improved antitumor activity and reduced toxicity.

Development of a Dual-Functional Hydrogel Using RGD and Anti-VEGF Aptamer.

Synthetic molecular libraries hold great potential to advance the biomaterial development. However, little effort is made to integrate molecules with molecular recognition abilities selected from different libraries into a single biomolecular material. The purpose of this work is to incorporate peptides and nucleic acid aptamers into a porous hydrogel to develop a dual-functional biomaterial. The data show that an anti-integrin peptide can promote the attachment and growth of endothelial cells in a 3D porous poly(ethylene glycol) hydrogel and an antivascular endothelial growth factor aptamer can sequester and release VEGF of high bioactivity. Importantly, the dual-functional porous hydrogel enhances the growth and survival of endothelial cells. This work demonstrates that molecules selected from different synthetic libraries can be integrated into one system for the development of novel biomaterials.

Enrichment of endogenous fractalkine and anti-inflammatory cells via aptamer-functionalized hydrogels.

Early recruitment of non-classical monocytes and their macrophage derivatives is associated with augmented tissue repair and improved integration of biomaterial constructs. A promising therapeutic approach to recruit these subpopulations is by elevating local concentrations of chemoattractants such as fractalkine (FKN, CX3CL1). However, delivering recombinant or purified proteins is not ideal due to their short half-lives, suboptimal efficacy, immunogenic potential, batch variabilities, and cost. Here we report an approach to enrich endogenous FKN, obviating the need for delivery of exogenous proteins. In this study, modified FKN-binding-aptamers are integrated with poly(ethylene glycol) diacrylate to form aptamer-functionalized hydrogels ("aptagels") that localize, dramatically enrich and passively release FKN in vitro for at least one week. Implantation in a mouse model of excisional skin injury demonstrates that aptagels enrich endogenous FKN and stimulate significant local increases in Ly6CloCX3CR1hi non-classical monocytes and CD206+ M2-like macrophages. The results demonstrate that orchestrators of inflammation can be manipulated without delivery of foreign proteins or cells and FKN-aptamer functionalized biomaterials may be a promising approach to recruit anti-inflammatory subpopulations to sites of injury. Aptagels are readily synthesized, highly customizable and could combine different aptamers to treat complex diseases in which regulation or enrichment of multiple proteins may be therapeutic.