Publications by authors named "Paolo Caliceti"

71 Publications

Pullulan Based Bioconjugates for Ocular Dexamethasone Delivery.

Pharmaceutics 2021 May 26;13(6). Epub 2021 May 26.

Faculty of Health Sciences, School of Pharmacy, University of Eastern Finland, Yliopistonranta 1C, 70211 Kuopio, Finland.

Posterior segment eye diseases are mostly related to retinal pathologies that require pharmacological treatments by invasive intravitreal injections. Reduction of frequent intravitreal administrations may be accomplished with delivery systems that provide sustained drug release. Pullulan-dexamethasone conjugates were developed to achieve prolonged intravitreal drug release. Accordingly, dexamethasone was conjugated to ~67 kDa pullulan through hydrazone bond, which was previously found to be slowly cleavable in the vitreous. Dynamic light scattering and transmission electron microscopy showed that the pullulan-dexamethasone containing 1:20 drug/glucose unit molar ratio (10% / dexamethasone) self-assembled into nanoparticles of 461 ± 30 nm and 402 ± 66 nm, respectively. The particles were fairly stable over 6 weeks in physiological buffer at 4, 25 and 37 °C, while in homogenized vitreous at 37 °C, the colloidal assemblies underwent size increase over time. The drug was released slowly in the vitreous and rapidly at pH 5.0 mimicking lysosomal conditions: 50% of the drug was released in about 2 weeks in the vitreous, and in 2 days at pH 5.0. In vitro studies with retinal pigment epithelial cell line (ARPE-19) showed no toxicity of the conjugates in the cells. Flow cytometry and confocal microscopy showed cellular association of the nanoparticles and intracellular endosomal localization. Overall, pullulan conjugates showed interesting features that may enable their successful use in intravitreal drug delivery.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3390/pharmaceutics13060791DOI Listing
May 2021

PEG-polyaminoacid based micelles for controlled release of doxorubicin: Rational design, safety and efficacy study.

J Control Release 2021 May 11;335:21-37. Epub 2021 May 11.

Department of Pharmaceutical and Pharmacological Sciences, University of Padova, via F. Marzolo 5, 35131 Padova, Italy.

A library of amphiphilic monomethoxypolyethylene glycol (mPEG) terminating polyaminoacid co-polymers able to self-assemble into colloidal systems was screened for the delivery and controlled release of doxorubicin (Doxo). mPEG-Glu/Leu random co-polymers were generated by Ring Opening Polymerization from 5 kDa mPEG-NH macroinitiator using 16:0:1, 8:8:1, 6:10:1, 4:12:1 γ-benzyl glutamic acid carboxy anhydride monomer/leucine N-carboxy anhydride monomer/PEG molar ratios. Glutamic acid was selected for chemical conjugation of Doxo, while leucine units were introduced in the composition of the polyaminoacid block as spacer between adjacent glutamic repeating units to minimize the steric hindrance that could impede the Doxo conjugation and to promote the polymer self-assembly by virtue of the aminoacid hydrophobicity. The benzyl ester protecting the γ-carboxyl group of glutamic acid was quantitatively displaced with hydrazine to yield mPEG-b-(hydGlu-r-Leu). Doxo was conjugated to the diblock co-polymers through pH-sensitive hydrazone bond. The Doxo derivatized co-polymers obtained with a 16:0:1, 8:8:1, 6:10:1 Glu/Leu/PEG ratios self-assembled into 30-40 nm spherical nanoparticles with neutral zeta-potential and CMC in the range of 4-7 μM. At pH 5.5, mimicking endosome environment, the carriers containing leucine showed a faster Doxo release than at pH 7.4, mimicking the blood conditions. Doxo-loaded colloidal formulations showed a dose dependent cytotoxicity on two cancer cell lines, CT26 murine colorectal carcinoma and 4T1 murine mammary carcinoma with IC slightly higher than those of free Doxo. The carrier assembled with the polymer containing 6:10:1 hydGlu/Leu/PEG molar ratio {mPEG-b-[(Doxo-hydGlu)-r-Leu]} was selected for subsequent in vitro and in vivo investigations. Confocal imaging on CT26 cell line showed that intracellular fate of the carrier involves a lysosomal trafficking pathway. The intratumor or intravenous injection to CT26 and 4T1 subcutaneous tumor bearing mice yielded higher antitumor activity compared to free Doxo. Furthermore, mPEG-b-[(Doxo-hydGlu)-r-Leu] displayed a better safety profile when compared to commercially available Caelyx®.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.jconrel.2021.05.010DOI Listing
May 2021

Screening of chemical linkers for development of pullulan bioconjugates for intravitreal ocular applications.

Eur J Pharm Sci 2021 Jun 2;161:105785. Epub 2021 Mar 2.

University of Padova, Department of Pharmaceutical and Pharmacological Sciences, Via F. Marzolo 5, 35131 Padova, Italy. Electronic address:

The treatment of posterior segment disorders of the eye requires therapeutic strategies to achieve drug effects over prolonged times. Innovative colloidal delivery systems can be designed to deliver drugs to the retina and prolong their intravitreal permanence. In order to exploit pullulan (Pull) as polymeric drug carrier for intravitreal drug delivery, derivatives of hydrophobic model molecule rhodamine B (RhB) were conjugated to the pullulan backbone through linkers with different stability, namely ether (Et), hydrazone (Hy) or ester (Es) bond to obtain Pull-Et-RhB, Pull-Hy-RhB and Pull-Es-RhB, respectively. Dynamic light scattering and transmission electron microscopy analyses showed that the polymer conjugates self-assembled into 20-25 nm particles. Pull-Et-RhB was fairly stable at all tested pH values. At the vitreal pH of 7.4, 50% of RhB was released from Pull-Hy-RhB and Pull-Es-RhB in 11 and 6 days, respectively. At endosomal pH (5.5), 50% of RhB was released from Pull-Hy-RhB and Pull-Es-RhB in 4 and 1 days, respectively. Multiple particle tracking analyses in ex vivo porcine eye model showed that the diffusivity of the bioconjugates in the vitreous was about 10 times lower than in water. Flow cytometry and confocal microscopy analyses showed that bioconjugates are remarkably taken up by the retinal RPE cells. In vivo studies showed that after intravitreal injection to mice, the bioconjugates localize in the ganglion cell layer and in the inner and outer plexiform layers. Pull-Hy-RhB particles were detected also inside the retinal blood vessels. These results demonstrate that pullulan with tailored linkers for drug conjugation is a promising vehicle for long-acting intravitreal injectables that are capable to permeate to the retina.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.ejps.2021.105785DOI Listing
June 2021

Glabrescione B delivery by self-assembling micelles efficiently inhibits tumor growth in preclinical models of Hedgehog-dependent medulloblastoma.

Cancer Lett 2021 02 26;499:220-231. Epub 2020 Nov 26.

Department of Molecular Medicine, University La Sapienza, Roma, Italy; Laboratory Affiliated to Istituto Pasteur Italia-Fondazione Cenci Bolognetti, Department of Molecular Medicine, University La Sapienza, Roma, Italy. Electronic address:

Aberrant activation of the Hedgehog (Hh) pathway leads to the development of several tumors, including medulloblastoma (MB), the most common pediatric brain malignancy. Hh inhibitors acting on GLI1, the final effector of Hh signaling, offer a valuable opportunity to overcome the pitfalls of the existing therapies to treat Hh-driven cancers. In this study, the toxicity, delivery, biodistribution, and anticancer efficacy of Glabrescione B (GlaB), a selective GLI1 inhibitor, were investigated in preclinical models of Hh-dependent MB. To overcome its poor water solubility, GlaB was formulated with a self-assembling amphiphilic polymer forming micelles, called mPEG-cholane. mPEG-cholane/GlaB showed high drug loading and stability, low cytotoxicity, and long permanence in the bloodstream. We found that mPEG-cholane efficiently enhanced the solubility of GlaB, thus avoiding the use of organic solvents. mPEG-cholane/GlaB possesses favorable pharmacokinetics and negligible toxicity. Remarkably, GlaB encapsulated in mPEG-cholane micelles was delivered through the blood-brain barrier and drastically inhibited tumor growth in both allograft and orthotopic models of Hh-dependent MB. Our findings reveal that mPEG-cholane/GlaB is a good candidate for the treatment of Hh-driven tumors and provide relevant implications for the translation of GlaB into clinical practice.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.canlet.2020.11.028DOI Listing
February 2021

Thermosensitive "Smart" Surfaces for Biorecognition Based Cell Adhesion and Controlled Detachment.

Macromol Biosci 2021 02 4;21(2):e2000277. Epub 2020 Nov 4.

Department of Pharmaceutical and Pharmacological Sciences, University of Padova, via F. Marzolo 5, Padova, 35131, Italy.

The biorecognition-based control of attachment/detachment of MCF-7 cancer cells from polymer-coated surfaces is demonstrated. A glass surface is coated with a thermoresponsive statistical copolymer of poly(N-isopropylacrylamide-co-acrylamide) [p(NIPAm-co-Am)], which is end-capped with the Gly-Arg-Gly-Asp-Ser (GRGDS) peptide, and the hydrophilic polymer poly(ethylene glycol) (PEG). Below the lower critical solution temperature (LCST) of p(NIPAm-co-Am) (38 °C), the copolymers are in the extended conformation, allowing for accessibility of the GRGDS peptides to membrane-associated integrins thus enabling cell attachment. Above the LCST, the p(NIPAm-co-Am) polymers collapse into globular conformations, resulting in the shielding of the GRGDS peptides into the PEG brush with consequent inaccessibility to cell-surface integrins, causing cell detachment. The surface coating is carried out by a multi-step procedure that included: glass surface amination with 3-aminopropyltriethoxysilane; reaction of mPEG -N-hydroxysuccinimide (NHS) and p(NIPam-co-Am) -bis-NHS with the surface aminopropyl groups and conjugation of GRGDS to the carboxylic acid termini of p(NIPam-co-Am) -COOH. A range of spectrophotometric, surface, and microscopy assays confirmed the identity of the polymer-coated substrates. Competition studies prove that MCF-7 cancer cells are attached via peptide recognition at the coated surfaces according to the mPEG /p(NIPam-co-Am) -GRGDS molar ratio. These data suggest the system can be exploited to modulate cell integrin/GRGDS binding for controlled cell capture and release.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/mabi.202000277DOI Listing
February 2021

From Conventional Therapies to Immunotherapy: Melanoma Treatment in Review.

Cancers (Basel) 2020 Oct 20;12(10). Epub 2020 Oct 20.

Department of Pharmaceutical and Pharmacological Sciences, University of Padova, Via F. Marzolo 5, 35131 Padova, Italy.

In this review, we discuss the use of oncolytic viruses and checkpoint inhibitors in cancer immunotherapy in melanoma, with a particular focus on combinatory therapies. Oncolytic viruses are promising and novel anti-cancer agents, currently under investigation in many clinical trials both as monotherapy and in combination with other therapeutics. They have shown the ability to exhibit synergistic anticancer activity with checkpoint inhibitors, chemotherapy, radiotherapy. A coupling between oncolytic viruses and checkpoint inhibitors is a well-accepted strategy for future cancer therapies. However, eradicating advanced cancers and tailoring the immune response for complete tumor clearance is an ongoing problem. Despite current advances in cancer research, monotherapy has shown limited efficacy against solid tumors. Therefore, current improvements in virus targeting, genetic modification, enhanced immunogenicity, improved oncolytic properties and combination strategies have a potential to widen the applications of immuno-oncology (IO) in cancer treatment. Here, we summarize the strategy of combinatory therapy with an oncolytic vector to combat melanoma and highlight the need to optimize current practices and improve clinical outcomes.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3390/cancers12103057DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7589099PMC
October 2020

Prospects of Replication-Deficient Adenovirus Based Vaccine Development against SARS-CoV-2.

Vaccines (Basel) 2020 Jun 10;8(2). Epub 2020 Jun 10.

Department of Virology, National Institute of Public Health-National Institute of Hygiene, Chocimska 24, 00-791 Warsaw, Poland.

The current appearance of the new SARS coronavirus 2 (SARS-CoV-2) and it quickly spreading across the world poses a global health emergency. The serious outbreak position is affecting people worldwide and requires rapid measures to be taken by healthcare systems and governments. Vaccinations represent the most effective strategy to prevent the epidemic of the virus and to further reduce morbidity and mortality with long-lasting effects. Nevertheless, currently there are no licensed vaccines for the novel coronaviruses. Researchers and clinicians from all over the world are advancing the development of a vaccine against novel human SARS-CoV-2 using various approaches. Herein, we aim to present and discuss the progress and prospects in the field of vaccine research towards SARS-CoV-2 using adenovirus (AdV) replication deficient-based strategies, with a comprehension that may support research and combat this recent world health emergency.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3390/vaccines8020293DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7349996PMC
June 2020

Recent Advances in Understanding the Protein Corona of Nanoparticles and in the Formulation of "Stealthy" Nanomaterials.

Front Bioeng Biotechnol 2020 3;8:166. Epub 2020 Apr 3.

First Surgical Clinic Section, Department of Surgical, Oncological and Gastroenterological Sciences, University of Padua, Padua, Italy.

In the last decades, the staggering progress in nanotechnology brought around a wide and heterogeneous range of nanoparticle-based platforms for the diagnosis and treatment of many diseases. Most of these systems are designed to be administered intravenously. This administration route allows the nanoparticles (NPs) to widely distribute in the body and reach deep organs without invasive techniques. When these nanovectors encounter the biological environment of systemic circulation, a dynamic interplay occurs between the circulating proteins and the NPs, themselves. The set of proteins that bind to the NP surface is referred to as the protein corona (PC). PC has a critical role in making the particles easily recognized by the innate immune system, causing their quick clearance by phagocytic cells located in organs such as the lungs, liver, and spleen. For the same reason, PC defines the immunogenicity of NPs by priming the immune response to them and, ultimately, their immunological toxicity. Furthermore, the protein corona can cause the physical destabilization and agglomeration of particles. These problems induced to consider the PC only as a biological barrier to overcome in order to achieve efficient NP-based targeting. This review will discuss the latest advances in the characterization of PC, development of stealthy NP formulations, as well as the manipulation and employment of PC as an alternative resource for prolonging NP half-life, as well as its use in diagnostic applications.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3389/fbioe.2020.00166DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7145938PMC
April 2020

Recellularized Colorectal Cancer Patient-derived Scaffolds as in vitro Pre-clinical 3D Model for Drug Screening.

Cancers (Basel) 2020 Mar 13;12(3). Epub 2020 Mar 13.

Fondazione Istituto di Ricerca Pediatrica Città della Speranza, 35129 Padua, Italy.

Colorectal cancer (CRC) shows highly ineffective therapeutic management. An urgent unmet need is the random assignment to adjuvant chemotherapy of high-risk stage II and stage III CRC patients without any predictive factor of efficacy. In the field of drug discovery, a critical step is the preclinical evaluation of drug cytotoxicity, efficacy, and efficiency. We proposed a patient-derived 3D preclinical model for drug evaluation that could mimic in vitro the patient's disease. Surgically resected CRC tissue and adjacent healthy colon mucosa were decellularized by a detergent-enzymatic treatment. Scaffolds were recellularized with HT29 and HCT116 cells. Qualitative and quantitative characterization of matched recellularized samples were evaluated through histology, immunofluorescences, scanning electron microscopy, and DNA amount quantification. A chemosensitivity test was performed using an increasing concentration of 5-fluorouracil (5FU). In vivo studies were carried out using zebrafish () animal model. Permeability test and drug absorption were also determined. The decellularization protocol allowed the preservation of the original structure and ultrastructure. Five days after recellularization with HT29 and HCT116 cell lines, the 3D CRC model exhibited reduced sensitivity to 5FU treatments compared with conventional 2D cultures. Calculated the half maximal inhibitory concentration (IC) for HT29 treated with 5FU resulted in 11.5 µM in 3D and 1.3 µM in 2D, and for HCT116, 9.87 µM in 3D and 1.7 µM in 2D. In xenograft experiments, HT29 extravasation was detected after 4 days post-injection, and we obtained a 5FU IC fully comparable to that observed in the 3D CRC model. Using confocal microscopy, we demonstrated that the drug diffused through the repopulated 3D CRC scaffolds and co-localized with the cell nuclei. The bioengineered CRC 3D model could be a reliable preclinical patient-specific platform to bridge the gap between in vitro and in vivo drug testing assays and provide effective cancer treatment.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3390/cancers12030681DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7140024PMC
March 2020

Dexamethasone Loaded Liposomes by Thin-Film Hydration and Microfluidic Procedures: Formulation Challenges.

Int J Mol Sci 2020 Feb 26;21(5). Epub 2020 Feb 26.

Department of Pharmaceutical and Pharmacological Sciences, University of Padova, Via F. Marzolo 5, 35131 Padova, Italy.

Liposomes have been one of the most exploited drug delivery systems in recent decades. However, their large-scale production with low batch-to-batch differences is a challenge for industry, which ultimately delays the clinical translation of new products. We have investigated the effects of formulation parameters on the colloidal and biopharmaceutical properties of liposomes generated with a thin-film hydration approach and microfluidic procedure. Dexamethasone hemisuccinate was remotely loaded into liposomes using a calcium acetate gradient. The liposomes produced by microfluidic techniques showed a unilamellar structure, while the liposomes produced by thin-film hydration were multilamellar. Under the same remote loading conditions, a higher loading capacity and efficiency were observed for the liposomes obtained by microfluidics, with low batch-to-batch differences. Both formulations released the drug for almost one month with the liposomes prepared by microfluidics showing a slightly higher drug release in the first two days. This behavior was ascribed to the different structure of the two liposome formulations. In vitro studies showed that both formulations are non-toxic, associate to human Adult Retinal Pigment Epithelial cell line-19 (ARPE-19) cells, and efficiently reduce inflammation, with the liposomes obtained by the microfluidic technique slightly outperforming. The results demonstrated that the microfluidic technique offers advantages to generate liposomal formulations for drug-controlled release with an enhanced biopharmaceutical profile and with scalability.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3390/ijms21051611DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7084920PMC
February 2020

In Vitro and in Vivo Behavior of Liposomes Decorated with PEGs with Different Chemical Features.

Mol Pharm 2020 Apr 21;17(4):1444. Epub 2020 Feb 21.

Department of Pharmaceutical and Pharmacological Sciences, University of Padova, via F. Marzolo 5, 35131 Padova, Italy.

View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1021/acs.molpharmaceut.0c00149DOI Listing
April 2020

In Vitro and in Vivo Behavior of Liposomes Decorated with PEGs with Different Chemical Features.

Mol Pharm 2020 02 22;17(2):472-487. Epub 2020 Jan 22.

Department of Pharmaceutical and Pharmacological Sciences , University of Padova , via F. Marzolo 5 , 35131 Padova , Italy.

The colloidal stability, in vitro toxicity, cell association, and in vivo pharmacokinetic behavior of liposomes decorated with monomethoxy-poly(ethylene glycol)-lipids (mPEG-lipids) with different chemical features were comparatively investigated. Structural differences of the mPEG-lipids used in the study included: (a) surface-anchoring moiety [1,2-distearoyl--glycero-3-phosphoethanolamine (DSPE), cholesterol (Chol), and cholane (Chln)]; (b) mPEG molecular weight (2 kDa mPEG and 5 kDa mPEG); and (c) mPEG shape (linear and branched PEG). In vitro results demonstrated that branched (mPEG)-DSPE confers the highest stealth properties to liposomes (∼31-fold lower cell association than naked liposomes) with respect to all PEGylating agents tested. However, the pharmacokinetic studies showed that the use of cholesterol as anchoring group yields PEGylated liposomes with longer permeance in the circulation and higher systemic bioavailability among the tested formulations. Liposomes decorated with mPEG-Chol had 3.2- and ∼2.1-fold higher area under curve (AUC) than naked liposomes and branched (mPEG)-DSPE-coated liposomes, respectively, which reflects the high stability of this coating agent. By comparing the PEGylating agents with same size, namely, linear 5 kDa PEG derivatives, linear mPEG-DSPE yielded coated liposomes with the best in vitro stealth performance. Nevertheless, the in vivo AUC of liposomes decorated with linear mPEG-DSPE was lower than that obtained with liposomes decorated with linear mPEG-Chol. Computational molecular dynamics modeling provided additional insights that complement the experimental results.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1021/acs.molpharmaceut.9b00887DOI Listing
February 2020

Nanovectors Design for Theranostic Applications in Colorectal Cancer.

J Oncol 2019 1;2019:2740923. Epub 2019 Oct 1.

First Surgical Clinic Section, Department of Surgical, Oncological and Gastroenterological Sciences, University of Padua, Via Nicolò Giustiniani 2, 35128 Padua, Italy.

Colorectal cancer (CRC) is a diffused disease with limited therapeutic options, none of which are often curative. Based on the molecular markers and targets expressed by the affected tissues, numerous novel approaches have been developed to study and treat this disease. In particular, the field of nanotechnology offers an astonishingly wide array of innovative nanovectors with high versatility and adaptability for both diagnosis and therapy (the so called "theranostic platforms"). However, such complexity can make the selection of a specific nanocarrier model to study a perplexing endeavour for the biomedical scientist or clinician not familiar with this field of inquiry. This review offers a comprehensive overview of this wide body of knowledge, in order to outline the essential requirements for the clinical viability evaluation of a nanovector model in CRC. In particular, the differences among the foremost designs, their specific advantages, and technological caveats will be treated, never forgetting the ultimate endpoint for these systems development: the clinical practice.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1155/2019/2740923DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6791220PMC
October 2019

Real-Time Label-Free Targeting Assessment and in Vitro Characterization of Curcumin-Loaded Poly-lactic--glycolic Acid Nanoparticles for Oral Colon Targeting.

ACS Omega 2019 Oct 1;4(16):16878-16890. Epub 2019 Oct 1.

Drug Research Program, Division of Pharmaceutical Biosciences, Faculty of Pharmacy, University of Helsinki, 00014 Helsinki, Finland.

The exploitation of curcumin for oral disease treatment is limited by its low solubility, poor bioavailability, and low stability. Surface-functionalized poly-lactic--glycolic acid (PLGA) nanoparticles (NPs) have shown promising results to ameliorate selective delivery of drugs to the gastro-intestinal tract. In this study, curcumin-loaded PLGA NPs (C-PLGA NPs) of about 200 nm were surface-coated with chitosan (CS) for gastro-intestinal mucosa adhesion, wheat germ agglutinin (WGA) for colon targeting or GE11 peptide for tumor colon targeting. Spectrometric and zeta potential analyses confirmed the successful functionalization of the C-PLGA NPs. Real-time label-free assessment of the cell membrane-NP interactions and NP cell uptake were performed by quartz crystal microbalance coupled with supported lipid bilayers and by surface plasmon resonance coupled with living cells. The study showed that CS-coated C-PLGA NPs interact with cells by the electrostatic mechanism, while both WGA- and GE11-coated C-PLGA NPs interact and are taken up by cells by specific active mechanisms. In vitro cell uptake studies corroborated the real-time label-free assessment by yielding a curcumin cell uptake of 7.3 ± 0.3, 13.5 ± 1.0, 27.3 ± 4.9, and 26.0 ± 1.3 μg per 10 HT-29 cells for noncoated, CS-, WGA-, and GE11-coated C-PLGA NPs, respectively. Finally, preliminary in vivo studies showed that the WGA-coated C-PLGA NPs efficiently accumulate in the colon after oral administration to healthy Balb/c mice. In summary, the WGA- and GE11-coated C-PLGA NPs displayed high potential for application as active targeted carriers for anticancer drug delivery to the colon.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1021/acsomega.9b02086DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6796886PMC
October 2019

Physical PEGylation to Prevent Insulin Fibrillation.

J Pharm Sci 2020 01 19;109(1):900-910. Epub 2019 Oct 19.

Department of Pharmaceutical and Pharmacological Sciences, University of Padova, via F. Marzolo 5, 35131 Padova, Italy.

Insulin is one of the most marketed therapeutic proteins worldwide. However, its formulation suffers from fibrillation, which affects the long-term storage limiting the development of novel devices for sustained delivery including portable infusion devices. We have investigated the effect of physical PEGylation on structural and colloidal stability of insulin by using 2 PEGylating agents terminating with polycyclic hydrophobic moieties, cholane and cholesterol: mPEG-cholane and mPEG-cholesterol, respectively. Microcalorimetric analyses showed that mPEG-cholane and mPEG-cholesterol efficiently bind insulin with binding constants (Ka) of 3.98 10 and 1.14 10 M, respectively. At room temperature, the 2 PEGylating agents yielded comparable structural stabilization of α-helix conformation and decreased dimerization of insulin. However, melting studies showed that mPEG-cholesterol has superior stabilizing effect of the protein conformation than mPEG-cholane. Furthermore, the fibrillation study showed that at a 1:1 and 1:5 insulin/polymer molar ratios, mPEG-cholesterol delays insulin fibrillation 40% and 26% more efficiently, respectively, as compared to mPEG-cholane which was confirmed by transmission electron microscopy imaging. Insulin was released from the mPEG-cholane and mPEG-cholesterol assemblies with comparable kinetic profiles. The physical PEGylation has a beneficial effect on the stabilization and shielding of the insulin structure into the monomeric form, which is not prone to fibrillation and aggregation.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.xphs.2019.10.020DOI Listing
January 2020

H-NMR metabolomics reveals the Glabrescione B exacerbation of glycolytic metabolism beside the cell growth inhibitory effect in glioma.

Cell Commun Signal 2019 08 28;17(1):108. Epub 2019 Aug 28.

IRCCS Neuromed, Pozzilli, IS, Italy.

Background: Glioma is the most common and primary brain tumors in adults. Despite the available multimodal therapies, glioma patients appear to have a poor prognosis. The Hedgehog (Hh) signaling is involved in tumorigenesis and emerged as a promising target for brain tumors. Glabrescione B (GlaB) has been recently identified as the first direct inhibitor of Gli1, the downstream effector of the pathway.

Methods: We established the overexpression of Gli1 in murine glioma cells (GL261) and GlaB effect on cell viability. We used H-nuclear magnetic resonance (NMR) metabolomic approach to obtain informative metabolic snapshots of GL261 cells acquired at different time points during GlaB treatment. The activation of AMP activated protein Kinase (AMPK) induced by GlaB was established by western blot. After the orthotopic GL261 cells injection in the right striatum of C57BL6 mice and the intranasal (IN) GlaB/mPEG-Cholane treatment, the tumor growth was evaluated. The High Performance Liquid Chromatography (HPLC) combined with Mass Spectrometry (MS) was used to quantify GlaB in brain extracts of treated mice.

Results: We found that GlaB affected the growth of murine glioma cells both in vitro and in vivo animal model. Using an untargeted H-NMR metabolomic approach, we found that GlaB stimulated the glycolytic metabolism in glioma, increasing lactate production. The high glycolytic rate could in part support the cytotoxic effects of GlaB, since the simultaneous blockade of lactate efflux with α-cyano-4-hydroxycinnamic acid (ACCA) affected glioma cell growth. According to the metabolomic data, we found that GlaB increased the phosphorylation of AMPK, a cellular energy sensor involved in the anabolic-to-catabolic transition.

Conclusions: Our results indicate that GlaB inhibits glioma cell growth and exacerbates Warburg effect, increasing lactate production. In addition, the simultaneous blockade of Gli1 and lactate efflux amplifies the anti-tumor effect in vivo, providing new potential therapeutic strategy for this brain tumor.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1186/s12964-019-0421-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6712882PMC
August 2019

Oligo-guanidyl targeted bioconjugates forming rod shaped polyplexes as a new nanoplatform for oligonucleotide delivery.

J Control Release 2019 09 7;310:58-73. Epub 2019 Aug 7.

Departement of Pharmaceutical and Pharmacological Sciences, University of Padova, Via F. Marzolo, 5, 35131 Padova, Italy.

Novel bioconjugates (Agm-M-PEG-FA) for active oligonucleotide (ON) delivery have been developed by conjugating a cationic oligo-guanidyl star-like shaped "head" (Agm-M) to a polymeric "tail" (PEG) terminating with folic acid (FA) as targeting agent or methoxy group (Agm-M-PEG-FA and Agm-M-PEG-OCH, respectively). Gel electrophoresis showed that the bioconjugates completely associated with ONs at 3 nitrogen/phosphate (N/P) ratio. Studies performed with folate receptor (FR)-overexpressing HeLa cells, showed that optimal cell up-take was obtained with the 75:25 w/w Agm-M-PEG-OCH:Agm-M-PEG-FA mixture. Dynamic light scattering and transmission electron microscopy showed that the polyplexes had size <80 nm with narrow polydispersity and rod-shaped morphology. The polyplexes were stable for several hours in plasma while ON was released in the presence of heparin concentration 16-times higher than the physiological one. The polyplexes displayed negligible cytotoxicity, hemolysis and low pro-inflammatory TNF-α release. Studies performed with FR-overexpressing HeLa and MDA-MB-231 cells using siRac1 revealed that the folated polyplexes caused significantly higher gene silencing (86.1 ± 9.6%) and inhibition of cell migration (40%) than the non-folated polyplexes obtained with Agm-M-PEG-OCH only. Although cytofluorimetric analyses showed similar cell uptake for both folated and non-folated polyplexes, confocal, TEM and competition studies showed that the folated polyplexes were taken-up by lysosome escaping caveolin-mediated pathway with final polyplex localization within cytosol, while non-folated polyplexes were preferentially taken-up via clathrin-mediated pathway to localize in the lysosomes. Finally, preliminary in vivo studies carried out in mice revealed that the folated polyplexes dispose in the tumor mass.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.jconrel.2019.08.005DOI Listing
September 2019

Novel Oligo-Guanidyl-PEG Carrier Forming Rod-Shaped Polyplexes.

Mol Pharm 2019 04 21;16(4):1678-1693. Epub 2019 Mar 21.

Department of Pharmaceutical and Pharmacological Sciences , University of Padova , Via F. Marzolo 5 35131 Padova , Italy.

A novel unconventional supramolecular oligo-cationic structure (Agm-M-PEG-OCH) has been synthesized to yield high efficiency therapeutic oligonucleotide (ON) delivery. Agm-M-PEG-OCH was obtained by a multistep protocol that included the conjugation of agmatine (Agm) moieties to maltotriose (M), which was further derivatized with one poly(ethylene glycol) (PEG) chain. Gel electrophoresis analysis showed that the 19 base pairs dsDNA model ON completely associates with Agm-M-PEG-OCH at 3 N/P molar ratio, which is in agreement with the in silico molecular predictions. Isothermal titration calorimetry (ITC) analyses showed that the Agm-M-PEG-OCH/ON association occurs through a combination of mechanisms depending on the N/P ratios resulting in different nanostructures. Dynamic light scattering (DLS) and transmission electron microscopy (TEM) revealed that the Agm-M-PEG-OCH/ON polyplexes have rod-shape structure with a mean diameter of 50-75 nm and aspect ratio depending on the N/P ratio. The polyplexes were stable over time in buffer, while a slight size increase was observed in the presence of serum proteins. Cell culture studies showed that neither Agm-M-PEG-OCH nor polyplexes displayed cytotoxic effects. Cellular uptake depended on the cell line and polyplex composition: cellular internalization was higher in the case of MCF-7 and KB cells compared to MC3T3-E1 cells and polyplexes with smaller aspect ratio were taken-up by cells more efficiently than polyplexes with higher aspect ratio. Finally, preliminary studies showed that our novel carrier efficiently delivered ONs into cells providing gene silencing.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1021/acs.molpharmaceut.9b00014DOI Listing
April 2019

Advances in Drug Delivery and Biomaterials: Facts and Vision.

Pharmaceutics 2019 Jan 21;11(1). Epub 2019 Jan 21.

Department of Drug Chemistry and Technologies, Sapienza University of Roma, P.le Aldo Moro 5, 00185 Roma, Italy.

Drug delivery and biomaterials are different fields of science but, at the same time, are tightly related and intertwined. The 2018 CRS Italy Chapter Annual Workshop aims to explore recent advances in design and development in these areas. Many colleagues from Europe participated to the Workshop, stimulating the discussion. To foster the discussion on recent research and networking opportunities, especially among younger attendees, all poster-presenting authors were asked to provide a short talk. The very friendly and stimulating atmosphere allowed the attendees to explore new frontiers and tackle new horizons.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3390/pharmaceutics11010048DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6359636PMC
January 2019

Control of Peptide Aggregation and Fibrillation by Physical PEGylation.

Biomacromolecules 2018 10 4;19(10):3958-3969. Epub 2018 Sep 4.

Department of Pharmaceutical and Pharmacological Sciences , Università degli Studi di Padova , via F. Marzolo 5 , 35131 Padova , Italy.

Peptide therapeutics have the potential to self-associate, leading to aggregation and fibrillation. Noncovalent PEGylation offers a strategy to improve their physical stability; an understanding of the behavior of the resulting polymer/peptide complexes is, however, required. In this study, we have performed a set of experiments with additional mechanistic insight provided by in silico simulations to characterize the molecular organization of these complexes. We used palmitoylated vasoactive intestinal peptide (VIP-palm) stabilized by methoxy-poly(ethylene glycol)-cholane (PEG-cholane) as our model system. Homogeneous supramolecular assemblies were found only when complexes of PEG-cholane/VIP-palm exceeded a molar ratio of 2:1; at and above this ratio, the simulations showed minimal exposure of VIP-palm to the solvent. Supramolecular assemblies formed, composed of, on average, 9-11 PEG-cholane/VIP-palm complexes with 2:1 stoichiometry. Our in silico results showed the structural content of the helical conformation in VIP-palm increases when it is complexed with the PEG-cholane molecule; this behavior becomes yet more pronounced when these complexes assemble into larger supramolecular assemblies. Our experimental results support this: the extent to which VIP-palm loses helical structure as a result of thermal denaturation was inversely related to the PEG-cholane:VIP-palm molar ratio. The addition of divalent buffer species and increasing the ionic strength of the solution both accelerate the formation of VIP-palm fibrils, which was partially and fully suppressed by 2 and >4 mol equivalents of PEG-cholane, respectively. We conclude that the relative freedom of the VIP-palm backbone to adopt nonhelical conformations is a key step in the aggregation pathway.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1021/acs.biomac.8b00887DOI Listing
October 2018

pH-Controlled Liposomes for Enhanced Cell Penetration in Tumor Environment.

ACS Appl Mater Interfaces 2018 May 15;10(21):17646-17661. Epub 2018 May 15.

Department of Pharmaceutical and Pharmacological Sciences , University of Padova , Via F. Marzolo 5 , Padova 35131 , Italy.

An innovative pH-switchable colloidal system that can be exploited for site-selective anticancer drug delivery has been generated by liposome decoration with a new novel synthetic non-peptidic oligo-arginine cell-penetration enhancer (CPE) and a quenching PEGylated counterpart that detaches from the vesicle surface under the acidic conditions of tumors. The CPE module ( Arg- DAG) is formed by four arginine units conjugated to a first-generation (G1) 2,2-bis(hydroxymethyl)propionic acid (bis-MPA)/2,2-bis(aminomethyl)propionic acid (bis-AMPA) polyester dendron terminating with 1,2-distearoyl-3-azidopropane for liposome bilayer insertion. The zeta potential of the Arg- DAG-decorated liposomes increased up to +32 mV as the Arg- DAG/lipids molar ratio increased. The Arg- DAG liposome shielding at pH 7.4 was provided by methoxy-PEG-polymethacryloyl sulfadimethoxine (mPEG-SDM) with 7.1 apparent p K. Zeta potential, surface plasmon resonance and synchrotron small-angle X-ray scattering analyses showed that at pH 7.4 mPEG-SDM associates with polycationic Arg- DAG-decorated liposomes yielding liposomes with neutral zeta potential. At pH 6.5, which mimics the tumor environment, mPEG-SDM detaches from the liposome surface yielding Arg- DAG exposure. Flow cytometry and confocal microscopy showed a 30-fold higher HeLa cancer cell association of the Arg- DAG-decorated liposomes compared to non-decorated liposomes. At pH 7.4, the mPEG-SDM-coated liposomes undergo low cell association while remarkable cell association occurred at pH 6.5, which allowed for the controlled intracellular delivery of model macromolecules and small molecules loaded in the liposome under tumor conditions.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1021/acsami.8b03469DOI Listing
May 2018

Switching of Macromolecular Ligand Display by Thermoresponsive Polymers Mediates Endocytosis of Multiconjugate Nanoparticles.

Bioconjug Chem 2018 04 14;29(4):1030-1046. Epub 2018 Mar 14.

School of Pharmacy , University of Nottingham , University Park , Nottingham NG72RD , United Kingdom.

Ligand-mediated targeting and internalization of plasma membrane receptors is central to cellular function. These types of receptors have accordingly been investigated as targets to facilitate entry of diagnostic and therapeutic constructs into cells. However, there remains a need to characterize how receptor targeting agents on nanoparticles interact at surface receptors and whether it is possible to control these interactions via exogenous stimuli. Here, we describe the switchable display of the iron-transporting protein, transferrin (Tf), at the surface of thermoresponsive polymer-coated gold nanoparticles and show that internalization of the coated nanoparticles into target cells changes across temperature ranges over which transferrin is expected to be sterically "hidden" by an extended polymer chain and then "revealed" by polymer chain collapse. The switching process is dependent on the numbers of transferrin molecules and thermoresponsive polymer chains attached and whether the assay temperature is above or below the transition temperatures of the responsive polymers at the nanoparticle surfaces. Significantly, however, the control of internalization is critically reliant on overall nanoparticle colloidal stability while the thermoresponsive component of the surface undergoes conformational change. The data show that the cell entry function of complex and large biomolecule ligands can be modulated by polymer-induced accessibility change but that a simple "hide and reveal" mechanism for ligand display following polymer chain collapse is insufficient to account for nanoparticle uptake and subsequent intracellular trafficking.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1021/acs.bioconjchem.7b00704DOI Listing
April 2018

New comprehensive studies of a gold(III) Dithiocarbamate complex with proven anticancer properties: Aqueous dissolution with cyclodextrins, pharmacokinetics and upstream inhibition of the ubiquitin-proteasome pathway.

Eur J Med Chem 2017 Sep 19;138:115-127. Epub 2017 Jun 19.

Università degli Studi di Padova, Dipartimento di Scienze Chimiche, Via F. Marzolo 1, 35131, Padova, Italy. Electronic address:

The gold(III)-dithiocarbamate complex AuL12 (dibromo [ethyl-N-(dithiocarboxy-kS,kS')-N-methylglycinate] gold(III)), is endowed with promising in vitro/in vivo antitumor activity and toxicological profile. Here, we report our recent strategies to improve its water solubility and stability under physiological conditions along with our efforts for unravelling its tangled mechanism of action. We used three types of α-cyclodextrins (CDs), namely β-CD, Me-β-CD and HP-β-CD to prepare aqueous solutions of AuL12. The ability of these natural oligosaccharide carriers to enhance water solubility of hydrophobic compounds, allowed drug stability of AuL12 to be investigated. Moreover, pharmacokinetic experiments were first carried out for a gold(III) coordination compound, after i.v. injection of the nanoformulation AuL12/HP-β-CD to female mice. The gold content in the blood samples was detected at scheduled times by AAS (atomic absorption spectrometry) analysis, highlighting a fast biodistribution with a t of few minutes and a slow escretion (t of 14.3 h). The in vitro cytotoxic activity of AuL12 was compared with the AuL12/HP-β-CD mixture against a panel of three human tumor cell lines (i.e., HeLa, KB and MCF7). Concerning the mechanism of action, we previously reported the proteasome-inhibitory activity of some our gold(III)-based compounds. In this work, we moved from the proteasome target to upstream of the important ubiquitin-proteasome pathway, testing the effects of AuL12 on the polyubiquitination reactions involving the Ub-activating (E1) and -conjugating (E2) enzymes.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.ejmech.2017.06.013DOI Listing
September 2017

Re-programming pullulan for targeting and controlled release of doxorubicin to the hepatocellular carcinoma cells.

Eur J Pharm Sci 2017 May 10;103:104-115. Epub 2017 Feb 10.

Department of Pharmaceutical & Pharmacological Sciences, University of Padova, Via F. Marzolo 5, 35131 Padova, Italy. Electronic address:

A novel bioconjugate for hepatocellular carcinoma (HCC) targeting was obtained by pullulan re-programming, which involves the backbone oxidation and conjugation of targeting peptide and doxorubicin (Doxo) through a releasable linker. Preliminary in vivo studies showed that the oxidation of 40 glucopyranose units (GPU) out of 100 remarkably reduced the pullulan unspecific liver tropism. This oxidized polymer was functionalized with PreS1 to selectively target the HCC and with rhodamine (Rhod) as label to perform in vitro cell up-take investigations. PreS1 and Rhod were conjugated to the aldehydes present along the oxidized pullulan backbone through a 3.4 and 2kDa PEG spacer, respectively, and by reductive amination. The resulting PreS1-Pull-Rhod contained a mean of 8 PreS1 per oxidized pullulan chain. Cell culture studies were performed by using HepG2/SERPINB3 cells that overexpress the serpine B3 receptor and control HepG2/EMPTY cells that do not overexpress the receptor. A comparative study by cytofluorimetry and confocal microscopy performed using PreS1-Pull-Rhod and Pull-Rhod (control polymer) showed that PreS1 conveys to the conjugate high cell selectivity. Afterwards, the oxidized pullulan was exploited to generate a targeted drug delivery system by conjugation of Doxo to the polymer backbone through a hydrazone pH-sensitive bond and NH-PEG-PreS1. The PreS1-Pull-Doxo conjugate showed a two-fold increase of anticancer activity with respect to the control Pull-Doxo towards HepG2/SERPINB3 cells.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.ejps.2017.02.016DOI Listing
May 2017

CNA-loaded PLGA nanoparticles improve humoral response againstS. aureus-mediated infections in a mouse model: subcutaneous vs. nasal administration strategy.

J Microencapsul 2016 Dec 12;33(8):750-762. Epub 2016 Dec 12.

a Department of Drug Sciences , University of Pavia , Pavia , Italy.

The aim of this work was the assessment of the "in vivo" immune response of a poly(lactide-co-glycolide)-based nanoparticulate adjuvant for a sub-unit vaccine, namely, a purified recombinant collagen-binding bacterial adhesion fragment (CNA19), against Staphylococcus aureus-mediated infections. "In vivo" immunogenicity studies were performed on mice: immunisation protocols encompassed subcutaneous and intranasal administration of CNA19 formulated as nanoparticles (NPs) and furthermore, CNA19-loaded NPs formulated in a set-up thermosetting chitosan-β-glycerolphosphate (chitosan-β-GP) solution for intranasal route in order to extend antigen exposure to nasal mucosa. CNA19 loaded NPs (mean size of about 195 nm, 9.04 ± 0.37μg/mg as CNA19 loading capacity) confirmed as suitable vaccine for subcutaneous administration with a more pronounced adjuvant effect (about 3-fold higher) with respect to aluminium, recognised as "reference" adjuvant. CNA19 loaded NPs formulated in an optimised thermogelling chitosan-β-GP solution showed promising results for eliciting an effective humoral response and a good chance as intranasal boosting dose.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1080/02652048.2016.1260661DOI Listing
December 2016

Enhanced selective sonosensitizing efficacy of ultrasound-based anticancer treatment by targeted gold nanoparticles.

Nanomedicine (Lond) 2016 Dec 15;11(23):3053-3070. Epub 2016 Sep 15.

Department of Drug Science & Technology, University of Torino, Via P. Giuria 13, 10125 Torino, Italy.

Aim: This study investigates cancer targeted gold nanoparticles as ultrasound sensitizers for the treatment of cancer.

Methods: The ultrasound sensitizer activity of folate-PEG decorated gold nanoparticles (FA-PEG-GNP) has been studied on human cancer cell lines that overexpress folate receptors (KB and HCT-116) and another that does not (MCF7), at two ultrasound energy densities (8 × 10 J cm and 8 × 10 J cm, for 5 min at 1.866 MHz).

Results: FA-PEG-GNP selectively targeted KB and HCT-116 cells and a remarkable reduction in cancer cell growth was observed upon ultrasound exposure, along with significant reactive oxygen species generation and increase in necrotic cells.

Conclusion: The combined use of targeting capacity and the ultrasound sensitizing effect, make FA-PEG-GNP promising candidates for the site-specific cancer treatment.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.2217/nnm-2016-0293DOI Listing
December 2016

In vitro and in vivo evaluation of an oral multiple-unit formulation for colonic delivery of insulin.

Eur J Pharm Biopharm 2016 Nov 9;108:76-82. Epub 2016 Aug 9.

Università degli Studi di Milano, Dipartimento di Scienze Farmaceutiche, Sezione di Tecnologia e Legislazione Farmaceutiche "M.E. Sangalli", Via G. Colombo 71, 20133 Milano, Italy. Electronic address:

A multiple-unit formulation for time-dependent colonic release of insulin was obtained by coating insulin and sodium glycocholate immediate-release minitablets with: (i) Methocel® E50, a low-viscosity hydroxypropyl methylcellulose (inner coating), (ii) 5:1 w/w Eudragit® NE/Explotab® V17, a mixture of a neutral polymethacrylate with a pore-forming superdisintegrant (intermediate coating), and (iii) Aqoat® AS, enteric-soluble hydroxypropyl methylcellulose acetate succinate (outer coating). Sodium glycocholate was added as a permeation enhancer while the inner, intermediate and outer coatings were aimed, respectively, at delaying the onset of release through swelling/erosion processes, extending the duration of the lag phase by slowing down water penetration into the underlying functional layer, and overcoming variable gastric residence time. In vitro studies showed that neither insulin nor sodium glycocholate were released from the three-layer system during 2h of testing in 0.1N HCl, while complete release of the protein and of the enhancer occurred in phosphate buffer, pH 6.8, after consistent lag phases. No significant changes were noticed in the release profiles following twelve-month storage at 4°C. Oral administration of the novel formulation to diabetic rats elicited a peak in the plasma insulin concentration after 6h, which was associated with a sharp decrease in the glycemic levels. The relative bioavailability and pharmacological availability of such a formulation, as determined vs. the uncoated tablets, were 2.2 and 10.3, respectively. Based on these results, the three-layer system presented was considered a potentially interesting tool for oral colonic delivery of insulin and adjuvant compounds.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.ejpb.2016.08.002DOI Listing
November 2016

A novel combined strategy for the physical PEGylation of polypeptides.

J Control Release 2016 Mar 7;226:35-46. Epub 2016 Feb 7.

Department of Pharmaceutical and Pharmacological Sciences, Università degli Studi di Padova, via F. Marzolo 5, 35131 Padova, Italy. Electronic address:

Poly(ethylene glycol) (PEG) may be covalently conjugated to peptide drugs to overcome their rapid clearance but in doing so their potency can be lost. Here, a non-covalent approach was used to conjugate PEG bearing a terminal cholanic moiety (mPEG5kDa-cholane) to a 28 amino acid peptide, vasoactive intestinal peptide (VIP). Palmitoylation of the peptide was essential to facilitate physical interaction via a single binding site involving two mPEG5kDa-cholane molecules with an affinity constant of ~3·10(4)M(-1); these calorimetry data corroborating Scatchard analysis of dissolution data. The peptide/polymer complex (below 10-12nm diameter) provided for up to 5000-fold greater solubility of the peptide at pH7.4 (4μg/mL) and markedly increased peptide solution stability at 25°C over 30days. Mannitol enabled the complex to be lyophilized to yield a freeze-dried formulation which was efficiently reconstituted albeit with an ~10% decrease in solubility. The predominantly α-helical conformation of the peptide alone at pH5-6.5 was lost at pH7.4 but fully recovered with 2 molar equivalents of mPEG5kDa-cholane. After lyophilization and reconstitution an ~10% loss of α-helical conformation was observed, which may reflect the equivalent decrease in solubility. Pharmacokinetic studies following subcutaneous administration of the peptide (0.1mg/Kg) alone and with 2 molar equivalents of polymer showed that mPEG5kDa-cholane dramatically increased peptide concentration in the systemic circulation. This is the first demonstration of non-covalent PEGylation of acylated peptides, an important biologic class, which improves in vitro and in vivo properties, and thereby may prove an alternative to covalent PEGylation strategies.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.jconrel.2016.02.009DOI Listing
March 2016

Design, Synthesis, and Photophysical Properties of Pyrroloquinoline-Based Compounds Showing Strong Blue Fluorescence as Potential Dyes for Biomedical Applications.

ChemMedChem 2015 Nov 8;10(11):1846-62. Epub 2015 Oct 8.

Department of Pharmaceutical and Pharmacological Sciences, University of Padova, 35131, Padova, Italy.

A small library of 3-ethylpyrrolo[3,2-f]quinoline derivatives was synthesized to identify a novel class of dyes for use in biological studies. According to the spectroscopic analyses performed to evaluate the fluorimetric parameters of quantum yield and brightness, 7-methyl- and 6,7-dimethylpyrroloquinolin(9)one derivatives were found to be the best blue luminescent dyes for biological applications. To enhance the luminescence profiles and to obtain probes that could be conjugated to functional groups of supramolecular drug delivery systems, these compounds were further modified at position 3 to obtain 3-heptanoic acid and 3-aminohexylpyrroloquinolin(9)one methylated derivatives. The most brilliant 6,7-dimethyl-3-aminohexylpyrroloquinolinone hydrochloride was conjugated to pullulan, a biocompatible polysaccharide used to produce colloidal systems for drug delivery. Comparative studies showed that this compound can be properly exploited as a blue fluorescent label in biological investigations, namely cell trafficking and pharmacokinetics/biodistribution studies. These molecules possess higher fluorescence efficiency than commercial dyes in biological media, making them suitable alternatives to commercially available products in current use.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/cmdc.201500366DOI Listing
November 2015