EGFR-Targeted Nanobody Functionalized Polymeric Micelles Loaded with mTHPC for Selective Photodynamic Therapy.

meta-Tetra(hydroxyphenyl)chlorin (mTHPC) is one of the most potent second-generation photosensitizers, clinically used for photodynamic therapy (PDT) of head and neck squamous cell carcinomas. However, improvements are still required concerning its present formulation (i.e., Foscan, a solution of mTHPC in ethanol/propylene glycol (40:60 w/w)), as mTHPC has the tendency to aggregate in aqueous media, e.g., biological fluids, and it has limited tumor specificity. In the present study, polymeric micelles with three different diameters (17, 24, and 45 nm) based on benzyl-poly(ε-caprolactone)-b-poly(ethylene glycol) (PCLn-PEG; n = 9, 15, or 23) were prepared with mTHPC loadings ranging from 0.5 to 10 wt % using a film-hydration method as advanced nanoformulations for this photosensitizer. To favor the uptake of the micelles by cancer cells that overexpress the epidermal growth factor receptor (EGFR), the micelles were decorated with an EGFR-targeted nanobody (named EGa1) through maleimide-thiol chemistry. The enhanced binding of the EGFR-targeted micelles at 4 °C to EGFR-overexpressing A431 cells, compared to low-EGFR-expressing HeLa cells, confirmed the specificity of the micelles. In addition, an enhanced uptake of mTHPC-loaded micelles by A431 cells was observed when these were decorated with the EGa1 nanobody, compared to nontargeted micelles. Both binding and uptake of targeted micelles were blocked by an excess of free EGa1 nanobody, demonstrating that these processes occur through EGFR. In line with this, mTHPC loaded in EGa1-conjugated PCL23-PEG (EGa1-P23) micelles demonstrated 4 times higher photocytotoxicity on A431 cells, compared to micelles lacking the nanobody. Importantly, EGa1-P23 micelles also showed selective PDT against A431 cells compared to the low-EGFR-expressing HeLa cells. Finally, an in vivo pharmacokinetic study shows that after intravenous injection, mTHPC incorporated in the P23 micelles displayed prolonged blood circulation kinetics, compared to free mTHPC, independently of the presence of EGa1. Thus, these results make these micelles a promising nanomedicine formulation for selective therapy.

An Olive Leaf Extract Rich in Polyphenols Promotes Apoptosis in Cervical Cancer Cells by Upregulating p21Cip/WAF1 Gene Expression.

Most of the common drugs used to treat the cervical cancer, which main etiological factor is the HPV infection, cause side effects and intrinsic/acquired resistance to chemotherapy. In this study we investigated whether an olive leaf extract (OLE), rich in polyphenols, was able to exert anti-tumor effects in human cervical cancer cells (HeLa). MTT assay results showed a reduction of HeLa cells viability OLE-induced, concomitantly with a gene and protein down-regulation of Cyclin-D1 and an up-regulation of p21, triggering intrinsic apoptosis. OLE reduced NFkB nuclear translocation, which constitutive activation, stimulated by HPV-oncoproteins, promotes cancer progression and functional studies revealed that OLE activated p21Cip/WAF1 in a transcriptional-dependent-manner, by reducing the nuclear recruitment of NFkB on its responsive elements. Furthermore, OLE treatment counteracted epithelial-to-mesenchymal-transition and inhibited anchorage-dependent and -independent cell growth EGF-induced. Finally, MTT assay results revealed that OLE plus Cisplatin strengthened the reduction of cells viability Cisplatin-induced, as OLE inhibited NFkB, AkT and MAPK pathways, all involved in Cisplatin chemoresistance. In conclusion, we demonstrated that in HeLa cells OLE exerts pro-apoptotic effects, elucidating the molecular mechanism and that OLE could mitigate Cisplatin chemoresistance. Further studies are needed to explore the potential coadiuvant use of OLE for cervical cancer treatment.

Molecularly Imprinted Microrods via Mesophase Polymerization.

The aim of the present research work was the synthesis of molecularly imprinted polymers (MIPs) with a rod-like geometry via "mesophase polymerization". The ternary lyotropic system consisting of sodium dodecyl sulfate (SDS), water, and decanol was chosen to prepare a hexagonal mesophase to direct the morphology of the synthesized imprinted polymers using theophylline, methacrylic acid, and ethylene glycol dimethacrylate as a drug model template, a functional monomer, and a crosslinker, respectively. The obtained molecularly imprinted microrods (MIMs) were assessed by performing binding experiments and in vitro release studies, and the obtained results highlighted good selective recognition abilities and sustained release properties. In conclusion, the adopted synthetic strategy involving a lyotropic mesophase system allows for the preparation of effective MIPs characterized by a rod-like morphology.

Sericin/Poly(ethylcyanoacrylate) Nanospheres by Interfacial Polymerization for Enhanced Bioefficacy of Fenofibrate: In Vitro and In Vivo Studies.

Fenofibrate is a lipophilic drug used in hypercholesterolemia and hypertriglyceridemia as a lipid-regulating agent; however, it is characterized by poor water solubility and low dissolution rate, which result in a low oral bioavailability. In the present study, sericin/poly(ethylcyanoacrylate) nanospheres are synthesized by interfacial polymerization in aqueous media and investigated as a novel sericin-based delivery system for improved and enhanced oral bioefficacy of fenofibrate. The incorporation of sericin into the prepared cyanoacrylate nanoparticles and their spherical shape are confirmed by Lowry assay and scanning electron microscopy, respectively. Hydrophilic and mucoadhesive properties of the synthesized nanospheres are also evaluated. Finally, both in vitro release and in vivo studies are performed and the oral absorbable amount of fenofibrate is calculated to be higher than 70% when incorporated into the polymeric material, reducing the levels of total cholesterol (TC), triacylglycerols (TG), very low-density lipoproteins (VLDL), and low-density lipoproteins (LDL) compared to fenofibrate alone.

Correlation between in vitro stability and pharmacokinetics of poly(ε-caprolactone)-based micelles loaded with a photosensitizer.

Polymeric micelles are extensively investigated as drug delivery systems for hydrophobic drugs including photosensitizers (PSs). In order to benefit from micelles as targeted delivery systems for PS, rather than only solubilizers, the stability and cargo retention of the (PS-loaded) micelles should be properly assessed in biologically relevant media to get insight into the essential parameters predicting their in vivo performance (i.e., pharmacokinetics). In the present study, asymmetric flow field-flow fractionation (AF4) was used to investigate the in vitro stability in human plasma of empty and meta-tetra(hydroxyphenyl)chlorin (mTHPC)-loaded dithiolane-crosslinked micelles based on poly(ɛ-caprolactone)-co-poly(1,2-dithiolane­carbonate)-b-poly(ethylene glycol) (p(CL-co-DTC)-PEG) and non (covalently)-crosslinked micelles composed of poly(ε-caprolactone)-b-poly(ethylene glycol) (pCL-PEG). AF4 allows separation of the micelles from plasma proteins, which showed that small non (covalently)-crosslinked pCL9-PEG (17 nm) and pCL15-PEG (22 nm) micelles had lower stability in plasma than pCL23-PEG micelles with larger size (43 nm) and higher degree of crystallinity of pCL, and had also lower stability than covalently crosslinked p(CL9-DTC3.9)-PEG and p(CL18-DTC7.5)-PEG micelles with similar small sizes (~20 nm). In addition, PS (re)distribution to specific plasma proteins was observed by AF4, giving strong indications for the (in)stability of PS-loaded micelles in plasma. Nevertheless, fluorescence spectroscopy in human plasma showed that the retention of mTHPC in non (covalently)-crosslinked but semi-crystalline pCL23-PEG micelles (>8 h) was much longer than that in covalently crosslinked p(CL18-DTC7.5)-PEG micelles (~4 h). In line with this, in vivo circulation kinetics showed that pCL23-PEG micelles loaded with mTHPC had significantly longer half-life values (t½-ß of micelles and mTHPC was 14 and 18 h, respectively) than covalently crosslinked p(CL18-DTC7.5)-PEG micelles (t½-ß of both micelles and mTHPC was ~2 h). As a consequence, long circulating pCL23-PEG micelles resulted in significantly higher tumor accumulation of both the micelles and loaded mTHPC as compared to short circulating p(CL18-DTC7.5)-PEG micelles. These in vivo data were in good agreement with the in vitro stability studies. In conclusion, the present study points out that AF4 and fluorescence spectroscopy are excellent tools to evaluate the (in)stability of nanoparticles in biological media and thus predict the (in)stability of drug loaded nanoparticles after i.v. administration, which is favorable to screen promising delivery systems with reduced experimental time and costs and without excessive use of animals.

Smart Bandage Based on Molecularly Imprinted Polymers (MIPs) for Diclofenac Controlled Release.

The aim of the present study was the development of a "smart bandage" for the topical administration of diclofenac, in the treatment of localized painful and inflammatory conditions, incorporating Molecularly Imprinted Polymers (MIPs) for the controlled release of this anti-inflammatory drug. For this purpose, MIP spherical particles were synthesized by precipitation polymerization, loaded with the therapeutic agent and incorporated into the bandage surface. Batch adsorption binding studies were performed to investigate the adsorption isotherms and kinetics and the selective recognition abilities of the synthesized MIP. In vitro diffusion studies were also carried out using Franz cells and the obtained results were reported as percentage of the diffused dose, cumulative amount of diffused drug, steady-state drug flux and permeability coefficient. Moreover, the biocompatibility of the developed device was evaluated using the EPISKIN™ model. The Scatchard analysis indicated that the prepared MIP is characterized by the presence of specific binding sites for diclofenac, which are not present in the corresponding non-imprinted polymer, and the obtained results confirmed both the ability of the prepared bandage to prolong the drug release and the absence of skin irritation reactions. Therefore, these results support the potential application of the developed "smart bandage" as topical device for diclofenac sustained release.

Interconnected PolymerS TeChnology (IPSTiC): An Effective Approach for the Modulation of 5α-Reductase Activity in Hair Loss Conditions.

Hair loss represents a condition that adversely affects the social life of patients. The most common cause is androgenetic alopecia (AGA), which is a genetically determined progressive hair-loss condition involving 5α-reductase. In this study, a novel anti-baldness agent based on Interconnected PolymerS TeChnology (IPSTiC), which is an effective strategy for the delivery of bioactive molecules, was developed. This product (IPSTiC patch hair) is based on a polymeric blend consisting of high molecular weight hyaluronic acid and soybean proteins and is able to improve efficacy and stability of bioactive ingredients such as Origanum vulgare leaf extract, Camellia Sinensis leaf extract, and Capsicum Annuum fruit extract. The efficacy of the developed anti-baldness agent was investigated by performing several tests including NO radical and 5α-reductase inhibition assays, stability studies under different conditions, and in vitro diffusion studies using Franz cells. The biocompatibility of IPSTiC patch hair was also evaluated by in vitro analysis of the pro-sensitising potential and EPISKIN model. The obtained results confirmed both the efficacy and safety of IPSTiC patch hair supporting the potential use of this product in the topical treatment of AGA.

Polymeric nanoparticle constructs as devices for antibacterial therapy.

Diseases related to bacterial infections represent a relevant challenge for public health. Despite the success obtained with the conventional antibiotic therapies, indeed, new drawbacks have been identified. In addition to poor drugs solubility and stability, adverse side effects, and many other factors which together lead to a low patient compliance, the antibiotic resistance and the lack in the development of new antimicrobial agents are the main problems. On the basis of these considerations, the research interest is focused on the exploration of new strategies able to circumvent these drawbacks improving the efficacy of current antibiotics. In this context, nanosized systems, which allow to enhance both the pharmacokinetic profiles and the mechanism of action of drugs, play a key role.

Synthesis of sericin-based conjugates by click chemistry: enhancement of sunitinib bioavailability and cell membrane permeation.

Sericin is a natural protein that has been used in biomedical and pharmaceutical fields as raw material for polypeptide-based drug delivery systems (DDSs). In this paper, it has been employed as pharmaceutical biopolymer for the production of sunitinib-polypeptide conjugate. The synthesis has been carried out by simple click reaction in water, using the redox couple l-ascorbic acid/hydrogen peroxide as a free radical grafting initiator. The bioconjugate molecular weight (50 kDa < Mw < 75 kDa) was obtained by SDS-PAGE, while the spectroscopic characteristics have been studied in order to reveal the presence of grafted sunitinib. In both FT-IR and UV/Vis spectra, signals corresponding to sunitinib functional groups have been identified. Since sunitinib is an anticancer drug characterized by low bioavailability and low permeability, the bioconjugation aimed at their enhancement. In vitro studies demonstrated that bioavailability has been increased to almost 74%, compared with commercial formulation. Also cell membrane permeability has been augmented in in vitro tests, in which membrane models have been used to determine the lipid membrane/physiological fluid partition coefficient (Kp). The log(Kp) value of the bioconjugate was increased to over 4. This effect resulted in a three-fold decrease of IC50 value against MCF-7 cells.

New insights for the use of quercetin analogs in cancer treatment.

AIM: Quercetin (Q1) is a flavonoid widely present in plants and endowed with several pharmacological properties mostly due to its antioxidant potential. Q1 shows anticancer activity and could be useful in cancer prevention. On the other hand, Q1 is poorly soluble in water and unstable in physiological systems, and its bioavailability is very low. METHODS: A small set of Q1 derivatives (Q2-Q9) has been synthesized following opportunely modified chemical procedures previously reported. Anticancer activity has been evaluated by MTT assay. Human Topoisomerases inhibition has been performed by direct enzymatic assays. Apoptosis has been evaluated by TUNEL assay. ROS production and scavenging activity have been determined by immunofluorescence. RESULTS: The anticancer profile of a small library of Q1 analogues, in which the OH groups were all or partially replaced with hydrophobic functional groups, has been evaluated. Two of the studied compounds demonstrated an interesting cytotoxic profile in two breast cancer models showing the capability to inhibit human Topoisomerases. CONCLUSION: The studied compounds represent suitable leads for the development of innovative anticancer drugs. [Formula: see text].

Biopolymeric self-assembled nanoparticles for enhanced antibacterial activity of Ag-based compounds.

Microbial infections still remain one of the main issues for human health. The rapid development of resistance towards the most common antimicrobial drugs in bacteria represents today a challenge in the infections management. In the present work we have investigated the antibacterial activity of a group of compounds, namely silver N-heterocyclic carbene complexes, against a broad spectrum of bacteria. For the most promising compound, a biopolymeric nanocarrier has been developed, in order to potentiate the metal complex activity against both Gram +ve and Gram -ve. The polymeric nanovehicle is based on dextran, modified with oleic acid residues, that confer amphiphilic properties to the polysaccharide. We have characterized the obtained biomaterial and studied its ability to self-assemble into nanoparticles in aqueous environment. Next, the transdermal diffusion analyses have been carried out to evaluate the ability of the polymeric particles to penetrate tissues. Thanks to the strategy adopted, we have fabricated an antibacterial system to which K. pneumoniae and E. coli are the most sensitive.

Calabrian Goji vs. Chinese Goji: A Comparative Study on Biological Properties.

Lycium barbarum (Goji) fruits are mainly cultivated in northwestern China and are well known for their beneficial and healthy effects. In this work, the biological and functional properties of Calabrian Goji extract, obtained from Goji berries cultivated in the Sibari Plain (in the Italian region of Calabria), were demonstrated. In order to evaluate the use of this extract as a food supplement for cognitive and mental disorders, the quantification of Carotenoids as Zeaxanthin equivalents was made. The antioxidant activity was investigated by evaluating the scavenging properties against 2,2'-diphenyl-1-picrylhydrazyl (DPPH) and 2,2'-Azinobis-(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) radicals and by performing the ORAC (Oxygen Radical Absorbance Capacity) assay. The inhibition of lipid peroxidation was quantified by bleaching test and the ability to inhibit acetylcholinesterase enzyme and to scavenge nitric oxide radical was also evaluated. All the results were compared to those obtained from a Chinese Goji extract used as a reference. Based on the reported data, Calabrian Goji might be used as a food supplement with a possible application in cognitive disorders, mental impairments and other neurodegenerative diseases, due to its biological properties and the high levels of Carotenoids.

Engineered Polymer-Based Nanomaterials for Diagnostic, Therapeutic and Theranostic Applications.

Nanomedicine can be defined as the medical application of molecular nanotechnology and it plays a key role and pharmaceutical research and development, especially related to cancer prevention, monitoring, diagnosis and treatment. In this context, nanomaterials are attracting significant research interest due to their abilities to stay in the blood for long time, accumulate in pathological sites including tumors or inflammatory areas via the enhanced permeability and retention (EPR) effect, and facilitate targeted delivery of specific therapeutic agents. In the last decades, considerable attention was attracted by the development of nano-sized carriers, based on natural or synthetic polymers, able to provide the controlled release of anticancer drugs in the aim to overcome the drawbacks associated to the conventional chemotherapy. Furthermore, when loaded with imaging agents, this kind of systems offers the opportunity to exploit optical or magnetic resonance imaging (MRI) in cancer diagnosis. Polymeric materials are characterized by several functionalities where both therapeutic and imaging components, and also targeting moieties, can be attached for simultaneous targeted therapy and imaging providing innovative platforms defined as theranostic agents with a great potential in monitoring and treatment of cancer.

Quercetin derivatives as novel antihypertensive agents: Synthesis and physiological characterization.

The antihypertensive flavonol quercetin (Q1) is endowed with a cardioprotective effect against myocardial ischemic damage. Q1 inhibits angiotensin converting enzyme activity, improves vascular relaxation, and decreases oxidative stress and gene expression. However, the clinical application of this flavonol is limited by its poor bioavailability and low stability in aqueous medium. In the aim to overcome these drawbacks and preserve the cardioprotective effects of quercetin, the present study reports on the preparation of five different Q1 analogs, in which all OH groups were replaced by hydrophobic functional moieties. Q1 derivatives have been synthesized by optimizing previously reported procedures and analyzed by spectroscopic analysis. The cardiovascular properties of the obtained compounds were also investigated in order to evaluate whether chemical modification affects their biological efficacy. The interaction with ß-adrenergic receptors was evaluated by molecular docking and the cardiovascular efficacy was investigated on the ex vivo Langendorff perfused rat heart. Furthermore, the bioavailability and the antihypertensive properties of the most active derivative were evaluated by in vitro studies and in vivo administration (1month) on spontaneously hypertensive rats (SHRs), respectively. Among all studied Q1 derivatives, only the ethyl derivative reduced left ventricular pressure (at 10(-8)M÷10(-6)M doses) and improved relaxation and coronary dilation. NOSs inhibition by L-NAME abolished inotropism, lusitropism and coronary effects. Chronic administration of high doses of this compound on SHR reduced systolic and diastolic pressure. Differently, the acetyl derivative induced negative inotropism and lusitropism (at 10(-10)M and 10(-8)÷10(-6)M doses), without affecting coronary pressure. Accordingly, docking studies suggested that these compounds bind both ß1/ß2-adrenergic receptors. Taking into consideration all the obtained results, the replacement of OH with ethyl groups seems to improve Q1 bioavailability and stability; therefore, the ethyl derivative could represent a good candidate for clinical use in hypertension.