Validation of an In Vitro Diagnostic Test for Endometriosis: Impact of Confounding Medical Conditions and Lesion Location.

With the aim to shorten the time for diagnosis and accelerate access to correct management, a non-invasive diagnostic test for endometriosis was developed and validated. The IVD test combines an ELISA test kit to quantify CA125 and BDNF concentrations in serum and a data treatment algorithm hosted in medical software processing results from the ELISA test and responses to six clinical variables. Serum samples and clinical variables extracted from psychometric questionnaires from 77 patients were collected from the Oxford Endometriosis CaRe Centre biobank (UK). Case/control classification was performed based on laparoscopy and histological verification of the excised lesions. Biomarkers serum concentrations and clinical variables were introduced to the software, which generates the qualitative diagnostic result ("positive" or "negative"). This test allowed the detection of 32% of cases with superficial endometriosis, which is an added value given the limited efficacy of existing imaging techniques. Even in the presence of various confounding medical conditions, the test maintained a specificity of 100%, supporting its suitability for use in patients with underlying medical conditions.

Development and Validation of an Endometriosis Diagnostic Method Based on Serum Biomarkers and Clinical Variables.

Endometriosis affects more than 10% of women of reproductive age, significantly impacting their quality of life. Diagnosis typically takes 4 to 11 years from symptom onset. The gold standard for diagnosing this disease, laparoscopy, is invasive, contributing to this delay in diagnosis. Two studies were conducted to develop a diagnostic test based on the combination of serum biomarkers and clinical variables. Study 1, the development study, aimed to: (i) confirm the ability of CA125, BDNF and clinical variables to differentiate between cases and controls, and (ii) develop a diagnostic algorithm based on these results. Study 2 validated the clinical performance of the developed in vitro diagnostic (IVD) test in diagnosing endometriosis. Serum samples and clinical variables extracted from psychometric questionnaires were obtained from the Oxford Endometriosis CaRe Centre biobank (UK). Case/control classification was performed based on laparoscopy and histological verification of the excised lesions. Studies 1 and 2 included n = 204 and n = 79 patients, respectively. Study 1 found a statistically significant difference between cases and controls for levels of both biomarkers. Of the assessed clinical variables from the patients' medical histories, six were found to be significantly different between endometriosis cases and controls. CA125, BDNF and these six clinical variables were combined into a multivariable prediction model. In Study 2, the IVD test demonstrated sensitivity and specificity values of 46.2% (25.5-66.8%) and 100% (86.7-100%), respectively. Due to its high specificity, this IVD test is a simple and accurate rule-in test for early disease identification, even in the presence of non-specific symptoms.

Intracellular responsive dual delivery by endosomolytic polyplexes carrying DNA anchored porous silicon nanoparticles.

Bioresponsive cytosolic nanobased multidelivery has been emerging as an enormously challenging novel concept due to the intrinsic protective barriers of the cells and hardly controllable performances of nanomaterials. Here, we present a new paradigm to advance nano-in-nano integration technology amenable to create multifunctional nanovehicles showing considerable promise to overcome restrictions of intracellular delivery, solve impediments of endosomal localization and aid effectual tracking of nanoparticles. A redox responsive intercalator chemistry comprised of cystine and 9-aminoacridine is designed as a cross-linker to cap carboxylated porous silicon nanoparticles with DNA. These intelligent nanocarriers are then encapsulated within novel one-pot electrostatically complexed nano-networks made of a zwitterionic amino acid (cysteine), an anionic bioadhesive polymer (poly(methyl vinyl ether-alt-maleic acid)) and a cationic endosomolytic polymer (polyethyleneimine). This combined nanocomposite is successfully tested for the co-delivery of hydrophobic (sorafenib) or hydrophilic (calcein) molecules loaded within the porous core, and an imaging agent covalently integrated into the polyplex shell by click chemistry. High loading capacity, low cyto- and hemo-toxicity, glutathione responsive on-command drug release, and superior cytosolic delivery are shown as achievable key features of the proposed formulation. Overall, formulating drug molecules, DNA and imaging agents, without any interference, in a physico-chemically optimized carrier may open a path towards broad applicability of these cost-effective multivalent nanocomposites for treating different diseases.

Microfluidics platform for glass capillaries and its application in droplet and nanoparticle fabrication.

The accessibility to microfluidics of a broader scientific community is often limited by the costly and complex manufacture of the chips. In this respect, we present a simple and reusable platform for the flexible and easy assembly of glass capillaries to create a microfluidics chip within minutes, with excellent chemical compatibility and durability, and without the need of using specialized infrastructure. To demonstrate the application of the proposed platform, we have used it to produce microparticles by the double emulsion approach, nanoparticles by nanoprecipitation, and screened the nanoparticles' size and polydispersity obtained upon modification of various parameters.

pH-Switch Nanoprecipitation of Polymeric Nanoparticles for Multimodal Cancer Targeting and Intracellular Triggered Delivery of Doxorubicin.

Theranostic nanoparticles are emerging as potent tools for noninvasive diagnosis, treatment, and monitoring of solid tumors. Herein, an advanced targeted and multistimuli responsive theranostic platform is presented for the intracellular triggered delivery of doxorubicin. The system consists of a polymeric-drug conjugate solid nanoparticle containing encapsulated superparamagnetic iron oxide nanoparticles (IO@PNP) and decorated with a tumor homing peptide, iRGD. The production of this nanosystem is based on a pH-switch nanoprecipitation method in organic-free solvents, making it ideal for biomedical applications. The nanosystem shows sufficient magnetization saturation for magnetically guided therapy along with reduced cytotoxicity and hemolytic effects. IO@PNP are largely internalized by endothelial and metastatic cancer cells and iRGD decorated IO@PNP moderately enhance their internalization into endothelial cells, while no enhancement is found for the metastatic cancer cells. Poly(ethylene glycol)-block-poly(histidine) with pH-responsive and proton-sponge properties promotes prompt lysosomal escape once the nanoparticles are endocyted. In addition, the polymer-doxorubicin conjugate solid nanoparticles show both intracellular lysosomal escape and efficient translocation of doxorubicin to the nuclei of the cells via cleavage of the amide bond. Overall, IO@PNP-doxorubicin and the iRGD decorated counterpart demonstrate to enhance the toxicity of doxorubicin in cancer cells by improving the intracellular delivery of the drug carried in the IO@PNP.

Microfluidic Assembly of a Multifunctional Tailorable Composite System Designed for Site Specific Combined Oral Delivery of Peptide Drugs.

Multifunctional tailorable composite systems, specifically designed for oral dual-delivery of a peptide (glucagon-like peptide-1) and an enzymatic inhibitor (dipeptidyl peptidase 4 (DPP4)), were assembled through the microfluidics technique. Both drugs were coloaded into these systems for a synergistic therapeutic effect. The systems were composed of chitosan and cell-penetrating peptide modified poly(lactide-co-glycolide) and porous silicon nanoparticles as nanomatrices, further encapsulated in an enteric hydroxypropylmethylcellulose acetylsuccinate polymer. The developed multifunctional systems were pH-sensitive, inherited by the enteric polymer, enabling the release of the nanoparticles only in the simulated intestinal conditions. Moreover, the encapsulation into this polymer prevented the degradation of the nanoparticles' modifications. These nanoparticles showed strong and higher interactions with the intestinal cells in comparison with the nonmodified ones. The presence of DPP4 inhibitor enhanced the peptide permeability across intestinal cell monolayers. Overall, this is a promising platform for simultaneously delivering two drugs from a single formulation. Through this approach peptides are expected to increase their bioavailability and efficiency in vivo both by their specific release at the intestinal level and also by the reduced enzymatic activity. The use of this platform, specifically in combination of the two antidiabetic drugs, has clinical potential for the therapy of type 2 diabetes mellitus.

Microfluidic assisted one-step fabrication of porous silicon@acetalated dextran nanocomposites for precisely controlled combination chemotherapy.

An advanced nanocomposite consisting of an encapsulated porous silicon (PSi) nanoparticle and an acid-degradable acetalated dextran (AcDX) matrix (nano-in-nano), was efficiently fabricated by a one-step microfluidic self-assembly approach. The obtained nano-in-nano PSi@AcDX composites showed improved surface smoothness, homogeneous size distribution, and considerably enhanced cytocompatibility. Furthermore, multiple drugs with different physicochemical properties have been simultaneously loaded into the nanocomposites with a ratiometric control. The release kinetics of all the payloads was predominantly controlled by the decomposition rate of the outer AcDX matrix. To facilitate the intracellular drug delivery, a nona-arginine cell-penetrating peptide (CPP) was chemically conjugated onto the surface of the nanocomposites by oxime click chemistry. Taking advantage of the significantly improved cell uptake, the proliferation of two breast cancer cell lines was markedly inhibited by the CPP-functionalized multidrug-loaded nanocomposites. Overall, this nano-in-nano PSi@polymer composite prepared by the microfluidic self-assembly approach is a universal platform for nanoparticles encapsulation and precisely controlled combination chemotherapy.

Fabrication of a multifunctional nano-in-micro drug delivery platform by microfluidic templated encapsulation of porous silicon in polymer matrix.

A multifunctional nano-in-micro drug delivery platform is developed by conjugating the porous silicon nanoparticles with mucoadhesive polymers and subsequent encapsulation into a pH-responsive polymer using microfluidics. The multistage platform shows monodisperse size distribution and pH-responsive payload release, and the released nanoparticles are mucoadhesive. Moreover, this platform is capable of simultaneously loading and releasing multidrugs with distinct properties.

Microfluidic assembly of monodisperse multistage pH-responsive polymer/porous silicon composites for precisely controlled multi-drug delivery.

We report an advanced drug delivery platform for combination chemotherapy by concurrently incorporating two different drugs into microcompoistes with ratiometric control over the loading degree. Atorvastatin and celecoxib were selected as model drugs due to their different physicochemical properties and synergetic effect on colorectal cancer prevention and inhibition. To be effective in colorectal cancer prevention and inhibition, the produced microcomposite contained hypromellose acetate succinate, which is insoluble in acidic conditions but highly dissolving at neutral or alkaline pH conditions. Taking advantage of the large pore volume of porous silicon (PSi), atorvastatin was firstly loaded into the PSi matrix, and then encapsulated into the pH-responsive polymer microparticles containing celecoxib by microfluidics in order to obtain multi-drug loaded polymer/PSi microcomposites. The prepared microcomposites showed monodisperse size distribution, multistage pH-response, precise ratiometric controlled loading degree towards the simultaneously loaded drug molecules, and tailored release kinetics of the loaded cargos. This attractive microcomposite platform protects the payloads from being released at low pH-values, and enhances their release at higher pH-values, which can be further used for colon cancer prevention and treatment. Overall, the pH-responsive polymer/PSi-based microcomposite can be used as a universal platform for the delivery of different drug molecules for combination therapy.

Microfluidic assembly of multistage porous silicon-lipid vesicles for controlled drug release.

A reliable microfluidic platform for the generation of stable and monodisperse multistage drug delivery systems is reported. A glass-capillary flow-focusing droplet generation device was used to encapsulate thermally hydrocarbonized porous silicon (PSi) microparticles into the aqueous cores of double emulsion drops, yielding the formation of a multistage PSi-lipid vesicle. This composite system enables a large loading capacity for hydrophobic drugs.

Microfluidic templated mesoporous silicon-solid lipid microcomposites for sustained drug delivery.

A major challenge for a drug-delivery system is to engineer stable drug carriers with excellent biocompatibility, monodisperse size, and controllable release profiles. In this study, we used a microfluidic technique to encapsulate thermally hydrocarbonized porous silicon (THCPSi) microparticles within solid lipid microparticles (SLMs) to overcome the drawbacks accompanied by THCPSi microparticles. Formulation and process factors, such as lipid matrixes, organic solvents, emulsifiers, and methods to evaporate the organic solvents, were all evaluated and optimized to prepare monodisperse stable SLMs. FTIR analysis together with confocal images showed the clear deposition of THCPSi microparticles inside the monodisperse SLM matrix. The formation of monodisperse THCPSi-solid lipid microcomposites (THCPSi-SLMCs) not only altered the surface hydrophobicity and morphology of THCPSi microparticles but also remarkably enhanced their cytocompatibility with intestinal (Caco-2 and HT-29) cancer cells. Regardless of the solubility of the loaded therapeutics (aqueous insoluble, fenofibrate and furosemide; aqueous soluble, methotrexate and ranitidine) and the pH values of the release media (1.2, 5.0, and 7.4), the time for the release of 50% of the payloads from THCPSi-SLMC was at least 1.3 times longer than that from the THCPSi microparticles. The sustained release of both water-soluble and -insoluble drugs together with a reduced burst-release effect from monodisperse THCPSi-SLMC was achieved, indicating the successful encapsulation of THCPSi microparticles into the SLM matrix. The fabricated THCPSi-SLMCs exhibited monodisperse spherical morphology, enhanced cytocompatibility, and prolonged both water-soluble and -insoluble drug release, which makes it an attractive controllable drug-delivery platform.

Co-delivery of a hydrophobic small molecule and a hydrophilic peptide by porous silicon nanoparticles.

Nanoparticulate drug delivery systems offer remarkable opportunities for clinical treatment. However, there are several challenges when they are employed to deliver multiple cargos/payloads, particularly concerning the synchronous delivery of small molecular weight drugs and relatively larger peptides. Since porous silicon (PSi) nanoparticles (NPs) can easily contain high payloads of drugs with various properties, we evaluated their carrier potential in multi-drug delivery for co-loading of the hydrophobic drug indomethacin and the hydrophilic human peptide YY3-36 (PYY3-36). Sequential loading of these two drugs into the PSi NPs enhanced the drug release rate of each drug and also their amount permeated across Caco-2 and Caco-2/HT29 cell monolayers. Regardless of the loading approach used, dual or single, the drug permeation profiles were in good correlation with their drug release behaviour. Furthermore, the permeation studies indicated the critical role of the mucus intestinal layer and the paracellular resistance in the permeation of the therapeutic compounds across the intestinal wall. Loading with PYY3-36 also greatly improved the cytocompatibility of the PSi NPs. Conformational analysis indicated that the PYY3-36 could still display biological activity after release from the PSi NPs and permeation across the intestinal cell monolayers. These results are the first demonstration of the promising potential of PSi NPs for simultaneous multi-drug delivery of both hydrophobic and hydrophilic compounds.