Biomimetic Ghost Nanomedicine-Based Optotheranostics for Cancer.

Theranostic medicine combines diagnostics and therapeutics, focusing on solid tumors at minimal doses. Optically activated photosensitizers are significant examples owing to their photophysical and chemical properties. Several optotheranostics have been tested that convert light to imaging signals, therapeutic radicals, and heat. Upon light exposure, conjugated photosensitizers kill tumor cells by producing reactive oxygen species and heat or by releasing cancer antigens. Despite clinical trials, these molecularly conjugated photosensitizers require protection from their surroundings and a localized direction for site-specific delivery during blood circulation. Therefore, cell membrane biomimetic ghosts have been proposed for precise and safe delivery of these optically active large molecules, which are clinically relevant because of their biocompatibility, long circulation time, bypass of immune cell recognition, and targeting ability. This review focuses on the role of biomimetic nanoparticles in the treatment and diagnosis of tumors through light-mediated diagnostics and therapy, providing insights into their preclinical and clinical status.

A Fluorinated BODIPY-Based Zirconium Metal-Organic Framework for In Vivo Enhanced Photodynamic Therapy.

Photodynamic therapy (PDT), an emergent noninvasive cancer treatment, is largely dependent on the presence of efficient photosensitizers (PSs) and a sufficient oxygen supply. However, the therapeutic efficacy of PSs is greatly compromised by poor solubility, aggregation tendency, and oxygen depletion within solid tumors during PDT in hypoxic microenvironments. Despite the potential of PS-based metal-organic frameworks (MOFs), addressing hypoxia remains challenging. Boron dipyrromethene (BODIPY) chromophores, with excellent photostability, have exhibited great potential in PDT and bioimaging. However, their practical application suffers from limited chemical stability under harsh MOF synthesis conditions. Herein, we report the synthesis of the first example of a Zr-based MOF, namely, 69-L2, exclusively constructed from the BODIPY-derived ligands via a single-crystal to single-crystal post-synthetic exchange, where a direct solvothermal method is not applicable. To increase the PDT performance in hypoxia, we modify 69-L2 with fluorinated phosphate-functionalized methoxy poly(ethylene glycol). The resulting 69-L2@F is an oxygen carrier, enabling tumor oxygenation and simultaneously acting as a PS for reactive oxygen species (ROS) generation under LED irradiation. We demonstrate that 69-L2@F has an enhanced PDT effect in triple-negative breast cancer MDA-MB-231 cells under both normoxia and hypoxia. Following positive results, we evaluated the in vivo activity of 69-L2@F with a hydrogel, enabling local therapy in a triple-negative breast cancer mice model and achieving exceptional antitumor efficacy in only 2 days. We envision BODIPY-based Zr-MOFs to provide a solution for hypoxia relief and maximize efficacy during in vivo PDT, offering new insights into the design of promising MOF-based PSs for hypoxic tumors.

Hydrogels for RNA delivery.

RNA-based therapeutics have shown tremendous promise in disease intervention at the genetic level, and some have been approved for clinical use, including the recent COVID-19 messenger RNA vaccines. The clinical success of RNA therapy is largely dependent on the use of chemical modification, ligand conjugation or non-viral nanoparticles to improve RNA stability and facilitate intracellular delivery. Unlike molecular-level or nanoscale approaches, macroscopic hydrogels are soft, water-swollen three-dimensional structures that possess remarkable features such as biodegradability, tunable physiochemical properties and injectability, and recently they have attracted enormous attention for use in RNA therapy. Specifically, hydrogels can be engineered to exert precise spatiotemporal control over the release of RNA therapeutics, potentially minimizing systemic toxicity and enhancing in vivo efficacy. This Review provides a comprehensive overview of hydrogel loading of RNAs and hydrogel design for controlled release, highlights their biomedical applications and offers our perspectives on the opportunities and challenges in this exciting field of RNA delivery.

Cathepsin B Processing Is Required for the In Vivo Efficacy of Albumin-Drug Conjugates.

Targeted drug delivery approaches that selectively and preferentially deliver therapeutic agents to specific tissues are of great interest for safer and more effective pharmaceutical treatments. We investigated whether cathepsin B cleavage of a valine-citrulline [VC(S)]-containing linker is required for the release of monomethyl auristatin E (MMAE) from albumin-drug conjugates. In this study, we used an engineered version of human serum albumin, Veltis High Binder II (HBII), which has enhanced binding to the neonatal Fc (fragment crystallizable) receptor (FcRn) to improve drug release upon binding and FcRn-mediated recycling. The linker-payload was conjugated to cysteine 34 of albumin using a carbonylacrylic (caa) reagent which produced homogeneous and plasma stable conjugates that retained FcRn binding. Two caa-linker-MMAE reagents were synthesized─one with a cleavable [VC(S)] linker and one with a noncleavable [VC(R)] linker─to question whether protease-mediated cleavage is needed for MMAE release. Our findings demonstrate that cathepsin B is required to achieve efficient and selective antitumor activity. The conjugates equipped with the cleavable [VC(S)] linker had potent antitumor activity in vivo facilitated by the release of free MMAE upon FcRn binding and internalization. In addition to the pronounced antitumor activity of the albumin conjugates in vivo, we also demonstrated their preferable tumor biodistribution and biocompatibility with no associated toxicity or side effects. These results suggest that the use of engineered albumins with high FcRn binding combined with protease cleavable linkers is an efficient strategy to target delivery of drugs to solid tumors.

'Passive' nanoparticles for organ-selective systemic delivery: design, mechanism and perspective.

Nanotechnology has shown tremendous success in the drug delivery field for more effective and safer therapy, and has recently enabled the clinical approval of RNA medicine, a new class of therapeutics. Various nanoparticle strategies have been developed to improve the systemic delivery of therapeutics, among which surface modification of targeting ligands on nanoparticles has been widely explored for 'active' delivery to a specific organ or diseased tissue. Meanwhile, compelling evidence has recently been reported that organ-selective targeting may also be achievable by systemic administration of nanoparticles without surface ligand modification. In this Review, we highlight this unique set of 'passive' nanoparticles and their compositions and mechanisms for organ-selective delivery. In particular, the lipid-based, polymer-based, and biomimetic nanoparticles with tropism to different specific organs after intravenous administration are summarized. The underlying mechanisms (e.g., protein corona and size effect) of these nanosystems for organ selectivity are also extensively discussed. We further provide perspectives on the opportunities and challenges in this exciting area of organ-selective systemic nanoparticle delivery.

Accurate and rapid microfluidic ELISA to monitor Infliximab titers in patients with inflammatory bowel diseases.

Inflammatory bowel disease (IBD) is a term used to describe disorders that involve chronic inflammation in the gastrointestinal tract, affecting more than 6.8 million people worldwide. Biological therapy is used in the most severe cases of IBD where anti-tumour necrosis factor-alpha (TNF-α) antibodies are the first choice for a biological treatment. When administrated to patients, these antibodies interact with TNF-α, usually overexpressed in these diseases, neutralizing its biological activity. Because of the chronic nature of these diseases, a recurring administration of the therapeutic antibodies is required, thus making therapy monitorization essential for the correct management of these diseases. The aim of this work is the development of an enzyme-linked immunosorbent assay (ELISA) microfluidic biosensor to quantify the therapeutic antibodies in IBD patient plasma samples, where the commercial monoclonal antibody Infliximab (IFX) is used as a model target. By providing a faster and more accurate measurement of IFX, the proposed method leads to improved therapy scheduling and a reduced risk of endogenous anti-drug antibodies (ADAs) reducing the efficacy of the treatment. The time needed between sample insertion and result output for the microfluidic ELISA (mELISA) is 24 minutes, drastically shorter than the time required by the conventional ELISA (cELISA). The mELISA presented in this work has a LoD of 0.026 µg mL-1, while commercially available solutions provide a LoD of 0.15 µg mL-1. Results acquired by the mELISA are highly correlated with the results obtained from the cELISA (r = 0.998; R2 = 0.996; p < 0.0001), demonstrating the validity of the microfluidic approach for the quantification of IFX from patient plasma and its potential for use at the point-of-care (POC).

Microfluidic device for multiplexed detection of fungal infection biomarkers in grape cultivars.

Early diagnosis of fungal infections, which have seen an increase due to different environmental factors, is essential to an appropriate treatment of the plant by avoiding proliferation of the pathogen without excessive fungicide applications. In this work, we propose a microfluidic based approach to a multiplexed, point-of-need detection system capable of identifying infected grape cultivars. The system relies on the simultaneous detection of three plant hormones: salicylic, azelaic and jasmonic acids with a total assay time under 7 minutes, with LODs of 15 µM, 10 µM and 4.4 nM respectively. The three detection assays are based on optical transduction, with the detection of salicylic and azelaic acids using transmission measurements, while the detection of jasmonic acid is a fluorescence-based assay. The molecular recognition event for each metabolite is different: nanoparticle conjugation for salicylic acid, enzymatic reaction for azelaic acid and antibody-antigen recognition for jasmonic acid. In this work, two cultivars, Trincadeira and Carignan, presented infections with two fungal pathogens, Botrytis cinerea and Erysiphe necator. The grapes were tested using the microfluidic system alongside the benchmark techniques such as, high-performance liquid chromatography and enzyme-linked immunosorbent assay. The microfluidic system was not only capable of distinguishing infected from healthy samples, but also capable of distinguishing between different infection types.

Platinum-Triggered Bond-Cleavage of Pentynoyl Amide and N-Propargyl Handles for Drug-Activation.

The ability to create ways to control drug activation at specific tissues while sparing healthy tissues remains a major challenge. The administration of exogenous target-specific triggers offers the potential for traceless release of active drugs on tumor sites from antibody-drug conjugates (ADCs) and caged prodrugs. We have developed a metal-mediated bond-cleavage reaction that uses platinum complexes [K2PtCl4 or Cisplatin (CisPt)] for drug activation. Key to the success of the reaction is a water-promoted activation process that triggers the reactivity of the platinum complexes. Under these conditions, the decaging of pentynoyl tertiary amides and N-propargyls occurs rapidly in aqueous systems. In cells, the protected analogues of cytotoxic drugs 5-fluorouracil (5-FU) and monomethyl auristatin E (MMAE) are partially activated by nontoxic amounts of platinum salts. Additionally, a noninternalizing ADC built with a pentynoyl traceless linker that features a tertiary amide protected MMAE was also decaged in the presence of platinum salts for extracellular drug release in cancer cells. Finally, CisPt-mediated prodrug activation of a propargyl derivative of 5-FU was shown in a colorectal zebrafish xenograft model that led to significant reductions in tumor size. Overall, our results reveal a new metal-based cleavable reaction that expands the application of platinum complexes beyond those in catalysis and cancer therapy.

Tetrazine Carbon Nanotubes for Pretargeted In†Vivo "Click-to-Release" Bioorthogonal Tumour Imaging.

The bioorthogonal inverse-electron-demand Diels-Alder (IEDDA) cleavage reaction between tetrazine and trans-cyclooctene (TCO) is a powerful way to control the release of bioactive agents and imaging probes. In this study, a pretargeted activation strategy using single-walled carbon nanotubes (SWCNTs) that bear tetrazines (TZ@SWCNTs) and a TCO-caged molecule was used to deliver active effector molecules. To optimize a turn-on signal by using in vivo fluorescence imaging, we developed a new fluorogenic near-infrared probe that can be activated by bioorthogonal chemistry and image tumours in mice by caging hemicyanine with TCO (tHCA). With our pretargeting strategy, we have shown selective doxorubicin prodrug activation and instantaneous fluorescence imaging in living cells. By combining a tHCA probe and a pretargeted bioorthogonal approach, real-time, non-invasive tumour visualization with a high target-to-background ratio was achieved in a xenograft mice tumour model. The combined advantages of enhanced stability, kinetics and biocompatibility, and the superior pharmacokinetics of tetrazine-functionalised SWCNTs could allow application of targeted bioorthogonal decaging approaches with minimal off-site activation of fluorophore/drug.

Microfluidic device for the point of need detection of a pathogen infection biomarker in grapes.

Bacterial, fungal and viral infections in plant systems are on the rise, most of which tend to spread quickly amongst crops. These pathogens are also gaining resistance to known treatments, which makes their early detection a priority to avoid extensive loss of crops and the spreading of disease to animal systems. In this work, we propose a microfluidic platform coupled with integrated thin-film silicon photosensors for the detection of pathogen infections in grapes. This detection was achieved by monitoring the concentration of Azelaic Acid (AzA). This small organic acid plays a significant role in the defense mechanism in plant systems. In this platform, the enzyme tyrosinase was immobilized on microbeads inside a microfluidic system. By colorimetric monitoring of the inhibitory effect of AzA on the enzyme tyrosinase in real time, it was possible, in under 10 minutes, to detect different concentrations of AzA in both buffer and spiked solutions of grape juice, in both cases with limits of detection in the 5-10 nM range. In addition, with this microfluidic device, it was possible to clearly distinguish infected from healthy grape samples at three different grape maturation points. Healthy grape samples showed AzA concentrations in the range of 10-20 nM (post-dilution) while infected samples have an estimated increase of AzA of 10-30×, results which were confirmed using HPLC. In both juice and grape samples an integrated sample preparation stage that decreases the phenol content of the solutions was required to achieve fit-for-purpose sensitivities to AzA.

Oral pH sensitive GNS@ab nanoprobes for targeted therapy of Helicobacter pylori without disturbance gut microbiome.

How to eradicate Helicobacter pylori (H. pylori) in vivo with antibiotic resistance owns tremendous clinical requirement. Herein, gold nanostars were conjugated with acid-sensitive cis-aconitic anhydride modified anti-H. pylori polyclonal antibodies, resultant pH sensitive gold nanostars@H. pylori-antibodies nanoprobes (GNS@Ab) were employed for the theranostics of H. pylori in vivo. Photoacoustic imaging confirmed that prepared GNS@Ab could target actively H. pylori in the stomach. GNS@Ab nanoprobes could kill H. pylori in vivo in model animals under NIR laser irradiation, all GNS@Ab nanoprobes could be excreted out of gut within 7 days after oral administration. Gastric local lesion caused by H. pylori restored to normal status within one month. GNS@Ab nanoprobes within therapeutic doses did not damage intestinal bacteria imbalance. Forty clinical specimens of H. pylori with antibiotic resistance were verified validity of GNS@Ab nanoprobes. Prepared oral pH-sensitive GNS@Ab nanoprobes own clinical translational potential in the theranostics of H. pylori in near future.

Designing Hydrogels for On-Demand Therapy.

Systemic administration of therapeutic agents has been the preferred approach to treat most pathological conditions, in particular for cancer therapy. This treatment modality is associated with side effects, off-target accumulation, toxicity, and rapid renal and hepatic clearance. Multiple efforts have focused on incorporating targeting moieties into systemic therapeutic vehicles to enhance retention and minimize clearance and side effects. However, only a small percentage of the nanoparticles administered systemically accumulate at the tumor site, leading to poor therapeutic efficacy. This has prompted researchers to call the status quo treatment regimen into question and to leverage new delivery materials and alternative administration routes to improve therapeutic outcomes. Recent approaches rely on the use of local delivery platforms that circumvent the hurdles of systemic delivery. Local administration allows delivery of higher "effective" doses while enhancing therapeutic molecules' stability, minimizing side effects, clearance, and accumulation in the liver and kidneys following systemic administration. Hydrogels have proven to be highly biocompatible materials that allow for versatile design to afford sensing and therapy at the same time. Hydrogels' chemical and physical versatility can be exploited to attain disease-triggered in situ assembly and hydrogel programmed degradation and consequent drug release, and hydrogels can also serve as a biocompatible depot for local delivery of stimuli-responsive therapeutic cargo. We will focus this Account on the hydrogel platform that we have developed in our lab, based on dendrimer amine and dextran aldehyde. This hydrogel is disease-responsive and capable of sensing the microenvironment and reacting in a graded manner to diverse pathologies to render different properties, including tissue adhesion, biocompatibility, hydrogel degradation, and embedded drug release profile. We also studied the degradation kinetics of our stimuli-responsive materials in vivo and analyzed the in vitro conditions under which in vitro-in vivo correlation is attained. Identifying key parameters in the in vivo microenvironment under healthy and disease conditions was key to attaining that correlation. The adhesive capacity of our dendrimer-dextran hydrogel makes it optimal for localized and sustained release of embedded drugs. We demonstrated that it affords the delivery of a range of therapeutics to combat cancer, including nucleic acids, small molecules, and antibody drugs. As a depot for local delivery, it allows a high dose of active biomolecules to be delivered directly at the tumor site. Immunotherapy, a recently blooming area in cancer therapy, may exploit stimuli-responsive hydrogels to impart systemic effects following localized therapy. Local delivery would enable release of the proper drug dose and improve drug bioavailability where needed at the same time creating memory and exerting the therapeutic effect systemically. This Account highlights our perspective on how local and systemic therapies provided by stimuli-responsive hydrogels should be used to impart more precise, long-lasting, and potent therapeutic outcomes.

Advances, challenges and opportunities for point-of-need screening of mycotoxins in foods and feeds.

The assurance of food and feed safety, including the identification and effective monitoring of multiple biological and chemical hazards, is a major societal challenge, given the increasing pace at which food commodities are demanded, produced and traded across the globe. Within this context, mycotoxins are globally widespread secondary fungal metabolites, which can contaminate crops either in the field or during storage and have serious human and animal health impacts such as carcinogenic, teratogenic and hepatotoxic effects. Therefore, their presence in a wide range of foods and feeds is strictly regulated, particularly in the European Union. In order to perform effective and routine monitoring of mycotoxin levels in the field prior to further processing, during transport or during processing, rapid, simple, portable and sensitive means of screening of regulated mycotoxins are in high demand. This review focuses on (1) discussing the relevance of mycotoxins and the standard approaches for their sampling and monitoring; and (2) compiling and discussing recent advances in miniaturized analytical tools for mycotoxin detection. This provides insights into current research efforts and opportunities to develop a truly integrated and fit-for-purpose analytical tool, suitable for use at critical points of the food, feed and raw material processing and distribution chains.

Implantable hydrogel embedded dark-gold nanoswitch as a theranostic probe to sense and overcome cancer multidrug resistance.

Multidrug resistance (MDR) in cancer cells is a substantial limitation to the success of chemotherapy. Here, we describe facile means to overcome resistance by silencing the multidrug resistance protein 1 (MRP1), before chemotherapeutic drug delivery in vivo with a single local application. Our platform contains hydrogel embedded with dark-gold nanoparticles modified with 5-fluorouracil (5-FU)-intercalated nanobeacons that serve as an ON/OFF molecular nanoswitch triggered by the increased MRP1 expression within the tumor tissue microenvironment. This nanoswitch can sense and overcome MDR prior to local drug release. The nanobeacons comprise a 5-FU intercalated DNA hairpin, which is labeled with a near-infrared (NIR) dye and a dark-quencher. The nanobeacons are designed to open and release the intercalated drug only upon hybridization of the DNA hairpin to a complementary target, an event that restores fluorescence emission due to nanobeacons conformational reorganization. Despite the cross-resistance to 5-FU, more than 90% tumor reduction is achieved in vivo in a triple-negative breast cancer model following 80% MRP1 silencing compared with the continuous tumor growth following only drug or nanobeacon administration. Our approach can be applied to reverse cross-resistance to other chemotherapeutic drugs and restore treatment efficacy. As a universal nanotheranostic probe, this platform can pave the way to early cancer detection and treatment.

Local triple-combination therapy results in tumour regression and prevents recurrence in a colon cancer model.

Conventional cancer therapies involve the systemic delivery of anticancer agents that neither discriminate between cancer and normal cells nor eliminate the risk of cancer recurrence. Here, we demonstrate that the combination of gene, drug and phototherapy delivered through a prophylactic hydrogel patch leads, in a colon cancer mouse model, to complete tumour remission when applied to non-resected tumours and to the absence of tumour recurrence when applied following tumour resection. The adhesive hydrogel patch enhanced the stability and provided local delivery of embedded nanoparticles. Spherical gold nanoparticles were used as a first wave of treatment to deliver siRNAs against Kras, a key oncogene driver, and rod-shaped gold nanoparticles mediated the conversion of near-infrared radiation into heat, causing the release of a chemotherapeutic as well as thermally induced cell damage. This local, triple-combination therapy can be adapted to other cancer cell types and to molecular targets associated with disease progression.

Self-assembled RNA-triple-helix hydrogel scaffold for microRNA modulation in the tumour microenvironment.

The therapeutic potential of miRNA (miR) in cancer is limited by the lack of efficient delivery vehicles. Here, we show that a self-assembled dual-colour RNA-triple-helix structure comprising two miRNAs-a miR mimic (tumour suppressor miRNA) and an antagomiR (oncomiR inhibitor)-provides outstanding capability to synergistically abrogate tumours. Conjugation of RNA triple helices to dendrimers allows the formation of stable triplex nanoparticles, which form an RNA-triple-helix adhesive scaffold upon interaction with dextran aldehyde, the latter able to chemically interact and adhere to natural tissue amines in the tumour. We also show that the self-assembled RNA-triple-helix conjugates remain functional in vitro and in vivo, and that they lead to nearly 90% levels of tumour shrinkage two weeks post-gel implantation in a triple-negative breast cancer mouse model. Our findings suggest that the RNA-triple-helix hydrogels can be used as an efficient anticancer platform to locally modulate the expression of endogenous miRs in cancer.

Plasmonic gold nanoparticles for detection of fungi and human cutaneous fungal infections.

Fungi, which are common in the environment, can cause a multitude of diseases. Warm, humid conditions allow fungi to grow and infect humans via the respiratory, digestive and reproductive tracts, genital area and other bodily interfaces. Fungi can be detected directly by microscopy, using the potassium hydroxide test, which is the gold standard and most popular method for fungal screening. However, this test requires trained personnel operating specialist equipment, including a fluorescent microscope and culture facilities. As most acutely infected patients seek medical attention within the first few days of symptoms, the optimal diagnostic test would be rapid and self-diagnostic simplifying and improving the therapeutic outcome. In suspensions of gold nanoparticles, Aspergillus niger can cause a colour change from red to blue within 2 min, as a result of changes in nanoparticle shape. A similar colour change was observed in the supernatant of samples of human toenails dispersed in water. Scanning electron microscopy, UV/Vis and Raman spectroscopy were employed to monitor the changes in morphology and surface plasmon resonance of the nanoparticles. The correlation of colour change with the fungal infection was analysed using the absorbance ratio at 520 nm/620 nm. We found a decrease in the ratio when the fungi concentration increased from 1 to 16 CFU/mL, with a detection limit of 10 CFU/mL. The test had an 80% sensitivity and a 95% specificity value for the diagnosis of athlete's foot in human patients. This plasmonic gold nanoparticle-based system for detection of fungal infections measures the change in shape of gold nanoparticles and generates coloured solutions with distinct tonality. Our application has the potential to contribute to self-diagnosis and hygiene control in laboratories/hospitals with fewer resources, just using the naked eye. Graphical abstract Colorimetric method for fungi detection with gold nano particles.

Empowering the Potential of Cell-Penetrating Peptides for Targeted Intracellular Delivery via Molecular Self-Assembly.

Cell-penetrating peptides (CPPs) have been widely explored as an effective tool to deliver a variety of molecules and nanoparticles into cells due to their intrinsic property to translocate across cell membranes. CPPs are easier to synthesize and functionalize, and their incorporation into delivery vehicles could be achieved by both non-covalent and covalent methods. Recent advances in molecular self-assembly have demonstrated the possibility to fabricate various nanostructures with precise control over the shape, size and presentation of diverse functionalities. Through rational design, CPPs could be used as a building block for the nanostructure formation via self-assembly, while providing the functionality for intracellular delivery. In this book chapter, we will describe strategies to design self-assembling CPP conjugates and illustrate how their self-assembled nanostructures are manipulated for effective intracellular delivery. Fundamental knowledge on CPPs and molecular self-assembly will also be described.

High-Throughput Nanoliter-Scale Analysis and Optimization of Multimodal Chromatography for the Capture of Monoclonal Antibodies.

Multimodal ligands are synthetic molecules comprising multiple types of interactions that have been increasingly used for the capture of different biopharmaceutical compounds within complex biological mixtures. For monoclonal antibodies (mAbs) in particular, these ligands have shown the possibility of direct capture from cell culture supernatants in native conditions, as well as enhanced selectivity and affinity compared to traditional single-mode ligands. However, performing the capture of a target mAb using multimodal chromatography comes with the need for extensive optimization of the operating conditions, due to the multitude of interactions that can be promoted in parallel. In this work, a high-throughput microfluidic platform was developed for the optimization of chromatographic conditions regarding the capture of an anti-interleukin 8 mAb, using a multimodal ligand (2-benzamido-4-mercaptobutanoic acid), under a wide range of buffer pH and conductivities. The interaction of the ligand with the fluorescently labeled target mAb was also analyzed with respect to the individual contribution of the hydrophobic (phenyl) and electrostatic (carboxyl) moieties using fluorescence microscopy. The results were further validated at the macroscale using prepacked columns in standard chromatography assays, and recovery yield values of 94.6% ± 5.2% and 97.7% ± 1.5% were obtained under optimal conditions for the miniaturized and conventional approaches, respectively. In summary, this study highlights that a microfluidic-based approach is a powerful analytical tool to expedite the optimization process while using reduced reagent volumes (<50 µL), less resin (∼70 nL), and delivering results in less than 1 min per assay condition.

Dual targeted immunotherapy via in vivo delivery of biohybrid RNAi-peptide nanoparticles to tumour-associated macrophages and cancer cells.

Lung cancer is associated with very poor prognosis and considered one of the leading causes of death worldwide. Here, we present highly potent and selective bio-hybrid RNAi-peptide nanoparticles that can induce specific and long-lasting gene therapy in inflammatory tumour associated macrophages (TAMs), via an immune modulation of the tumour milieu combined with tumour suppressor effects. Our data prove that passive gene silencing can be achieved in cancer cells using regular RNAi NPs. When combined with M2 peptide-based targeted immunotherapy that immuno-modulates TAMs cell-population, a synergistic effect and long-lived tumour eradication can be observed along with increased mice survival. Treatment with low doses of siRNA (ED50 0.0025-0.01 mg/kg) in a multi and long-term dosing system substantially reduced the recruitment of inflammatory TAMs in lung tumour tissue, reduced tumour size (∼95%) and increased animal survival (∼75%) in mice. Our results suggest that it is likely that the combination of silencing important genes in tumour cells and in their supporting immune cells in the tumour microenvironment, such as TAMs, will greatly improve cancer clinical outcomes.