The Pediatric Committee at the European Medicines Agency identified the needs of the development of age-appropriate formulation of captopril in pediatric population for the treatment of cardiovascular diseases and diabetic nephropathy. Captopril (CAT) is currently administered by extemporaneous liquid formulation or tablet due to its limited water stability. Therefore, polymeric nanoparticles were developed for transdermal delivery of CAT for obtaining a prolonged CAT release as well as an easy dosage control with high compliance of pediatric patients. Cellulose acetate phthalate (CAP) and chitosan (CH) were chosen to prepare nanoparticles by nanoprecipitation method-dropping technique without using surfactants. CAP nanoparticles and CAP nanoparticles combined with CH in different concentrations (1:1 w/w and 1:3 w/w) were produced both unloaded and loaded with CAT. Nanoparticles were characterized in terms of size, drug loading efficiency and physical stability during the time (1-28 days). Chemical stability of drug in the dispersion was investigated. Results show that CAP nanoparticles have no drug loading capacity, whereas CH allows the encapsulation of CAT; highest drug loading is obtained when 1:3 CAP:CH w/w ratio was used (64.6±7.6%). The particle preparation at 60°C enhances the interaction of CAT with the polymer matrix. The size of loaded CAP nanoparticles is 515.6±5.2 nm, whereas that of CAP-CH nanoparticles is 279.8±2.5 nm (1:1 w/w ratio) and 408.1±9.5 nm (1:1 w/w ratio) with a PDI values around 0.2, resulting in a homogeneous system. Good physical stability of all formulations during the time is observed. At the moment, CAT appears stable in the dispersions. In conclusion, CAP-CH nanoparticles prepared using a 1:3 w/w ratio show good properties for developing suitable formulation for transdermal delivery of CAT.

The stilbenes are bioactive molecules with a big amount of health benefits and many possibilities in pharmaceutical industry. Several authors have reported a high number of properties for these compounds, including anticancer, antioxidant, anti-inflammatory, antidiabetic, neuroprotective or antimicrobial activities. However, their low aqueous solubility and their ease degradation could lead to low concentration of bioactive compound in the target tissue. For this reason, in the present study, the inclusion complexes of oxyresveratrol with α-, β-, and γ- cyclodextrins are characterized using DSC, TGA, SEM and molecular docking in order to increase the stability of the molecule. All these techniques showed that β-cyclodextrin (β-CD) forms the best complexes.

The stability of oxyresveratrol and oxyresveratrol/ β-CD complexes in different aqueous solutions was evaluated by measuring ºBrix, pH and UV-Vis spectra. The effect of encapsulation on the solubility and antioxidant activity of oxyresveratrol was also analysed. The results indicated that solutions were stable for at least five weeks, especially when stored in darkness, and that cyclodextrin supplementation leads to a higher concentration and antioxidant capacity of the solubilized bioactive compound than when it is not used. These results and the increase in antioxidant activity could be interesting for the pharmaceutical industry and for drugs enriched in oxyresveratrol.

Neochlorogenic acid, a less-studied caffeoylquinic acid, isomer of chlorogenic acid, has been seen to possess antioxidant, antifungal, anti-inflammatory and anticarcinogenic effects, which makes it an interesting bioactive compound for incorporation in drugs, nutraceuticals or functional foods. However, its poor solubility in water and susceptibility to oxidation make such a task difficult. To overcome that, its encapsulation in cyclodextrins (CDs) is proposed. The fluorescence of neochlorogenic acid in different pH conditions was analyzed, and caffeic acid was proved to be the fluorescent moiety in the molecule. An encapsulation model whereby the ligand poses two potential complexation sites (caffeic and D-(-)-quinic moieties), showed that α-CD and HP-β-CD formed the best inclusion complexes with neochlorogenic acid, followed by M-β-CD, β-CD and γ-CD. Molecular docking with the two best CDs gave better scores for α-CD, despite HP-β-CD providing stabilization through H-bonds. The encapsulation of chlorogenic acid led to a similar CD order and scores, although constants were higher for α-CD, β-CD and M-β-CD, lower for HP-β-CD, and negligible for γ-CD. The solubility and the susceptibility to oxidation of neochlorogenic acid improved after complexation with α-CD and HP-β-CD, while the antioxidant activity of both isomers was maintained. These results could lead to obtaining more stable inclusion complexes with caffeoylquinic acids for applications in the pharmaceutical industry.

Introduction and Objectives: The use of cyclodextrins in ophthalmic formulations have great potential due to their ability to improve drug solubility by forming inclusion complexes, increasing drug bioavailability and stability. Different safety levels have been described at ophthalmic level for different types of cyclodextrins [1,2]. The aim of this work was to study possible corneal alterations after administration of cyclodextrin aqueous solutions: α-cyclodextrin, β-cyclodextrin, γ-cyclodextrin, hydroxypropyl-α-cyclodextrin, hydroxypropyl-β-cyclodextrin, hydroxypropyl-γ-cyclodextrin, partially methylated-β-cyclodextrin and sulfobutylether-β-cyclodextrin. In addition, the in vitro and in vivo bioadhesive behavior of cyclodextrins has also compared.

Methods: BCOP trial was performed with fresh bovine corneas. Corneal transparency and fluorescein permeability were measured before of contact with the cyclodextrin solutions. HET-CAM assay was performed to quantify the irritation potential. Corneal Mucoadhesive Assay was performed by SHIMADZU^® texturometer using fresh bovine corneas. Ophthalmic permanence studies by PET were carried out in rats. The formulations labelled with ¹⁸F-FDG, were instilled into the eye and static PET frames at different times were acquired.

Results: BCOP: Partially methylated-β-cyclodextrin shows transparency values similar to those of the positive control. The fluorescein permeability has been affected to a greater extent by the solution of α-cyclodextrin and hydroxypropyl-γ-cyclodextrin. HET-CAM: None of the formulations caused damage to the CAM blood vessels except αCD which modified the vessel´s appearance. Corneal Mucoadhesive Assay: All cyclodextrin solutions have similar bioadhesive properties, except for SBECD that presents a significantly lower bioadhesion work-value compared to the other cyclodextrins. PET: Hydroxypropylic cyclodextrins have a better t_1/2 on the ocular surface compared to the other tested cyclodextrins.

Conclusion: All cyclodextrins were safe with the exception of partially methylated-β-cyclodextrin. Hydroxypropylic cyclodextrins improve the ocular surface permanence so they could be useful for the preparation of ophthalmic formulations as long as they are the most suitable for the generation of inclusion complexes with the desired drug.

References

[1] EMA, Background Review for Cyclodextrins Used as Excipients, pp. 1–17, 2014.

[2] Marc Francois,1 Eric Snoeckx,1 Peter Putteman,1 Fons Wouters,1 Eddy De Proost,1 Urbain Delaet,1 Jef Peeters,2and Marcus E. Brewster. A mucoadhesive, cyclodextrin-based vaginal cream formulation of itraconazole. AAPS PharmSci. 2003;5(1):E5.

Acknowlwdgements

X.G-O and R.V-F acknowledges the financial support of Health Research Institute Foundation of Santiago de Compostela (FIDIS).

Electrospinning is a powerful and cost-effective method that applies a strong electric field and polymer solution to fabricate versatile nanofiber using a wide array of raw materials for different biological applications. Electrospun nanofibers have developed as potential materials for the manufacturing of nanocarriers for therapeutic platforms due to controllable surface decoration, large surface areas, complex pore structure, good surface functionalization with high biocompatibility. The prominence of this technique is the possibility of merging low soluble drugs loaded in the nanofibers to enhance drug release and bioavailability. Incorporation of metal nanomaterials with electrocatalytic and/or plasmonic features into electrospun polymer nanofiber enhances thermodynamic and mechanical properties to obtain synergetic function and properties. This review discussed an overview on the theory of electrospinning, achievements and concerns, and challenges currently face in fabricating an efficient drug delivery system. Additionally, the drug-loaded into electrospun nanocarriers for drug delivery applications and opportunities, and insights into future prospects were explained.

Introduction and objectives: Noninfectious uveitis is a disease often caused by an autoimmune response, inflammatory cytokines promote the activation of T-cells and trigger recruitment of large numbers of circulation inflammatory leukocytes into the eye. This process may cause irreversible tissue damage and visual impairment. Since tacrolimus inhibits T-cell proliferation and suppresses the release of inflammatory cytokines, it can theoretically be used to reduce inflammatory activity in uveitis patients [1]. Hospital pharmacy prepares tacrolimus eye drops reformulating from parenteral drugs (Prograf^®) as magistral formulations because there is not any commercial alternative. However, Prograf^® (tacrolimus solubilized in ethanol) has some irritating compounds that cause discomfort and unpleasantness to the patient, so these excipients had to be removed. Due to tacrolimus poorly solubility, the purpose of this work was to improve the drug solubility complexing tacrolimus with HPßCD, evaluate the safety, and study the ocular permanence of the eye drops.

Materials and Methods: Initially, tacrolimus phase solubility diagram in water with HPßCD was performed. It was studied the tacrolimus solubilization comparing the interaction between tacrolimus-HPßCD complex using water (MilliQ^®), BSS^® (Balanced Salt Solution, Alcon) and Liquifilm^® (Allergan) as vehicles. Irritation Ocular Assay (HET-CAM) and BCOP studies were performed in order to evaluate the safety of the formulations. In vivo PET/CT studies were carried out on anesthetized male Sprague-Dawley rats. 7.5 μL of each formulation labelled with ¹⁸F-fluorodeoxyglucose (¹⁸F-FDG) was instilled into the eye and PET/CT images were acquired at different times (0,30,75, 120, 240 and 300 min post-administration). Two animals (4 eyes) were tested for each formulation.

Results and Discussion: The solubility diagram in water was A_P type and stability constant values of K_1:1=148.2 mol^-1 and K_1:2=1.406 mol^-1. It can be seen a better tacrolimus solubility when it is combined the HPßCD and Liquifilm^® this may be because this vehicle incorporates polyvinyl alcohol (PVA), a polymer that has demonstrated a synergistic solubilization effect when it is combined with HPßCD [2]. HET-CAM assay and BCOP did not show blood vessels injury and any considerable change in the corneal surface. In vivo ocular permanence shows that formulations containing higher HPßCD concentrations give a better ocular surface permanence.

Conclusion: Results reveal tacrolimus solubility improvement, irritation absence on the ocular surface and a prolonged permanence time over the eye. These changes in the formulation compounds would enhance the patient’s adherence-to-treatment, reducing their eye discomfort.

Acknowledgements: X.G-O and R.V-F acknowledge the financial support of Health Research Institute Foundation of Santiago de Compostela (FIDIS).

References:

[1]. Zhai J, Gu J, Yuan J, Chen J. Tacrolimus in the treatment of ocular diseases. 2011;25:89–103.

[2]. Saokham, P.; Muankaew, C.; Jansook, P.; Loftsson, T. Solubility of Cyclodextrins and Drug/Cyclodextrin Complexes. Molecules2018, 23, 1161.

In this project, we report the synthesis of well-defined charged polymers with narrow molecular weight distribution using Reversible Addition Fragmentation chain Transfer (RAFT) polymerisation. Block-copolymers were obtained by sequential RAFT polymerisation. Optimal control in RAFT polymerisation was confirmed by the linear increase of molecular weight and the low PDI of the polymers (<1.2), as determined by ¹H NMR analysis and GPC, respectively. Spherical sub-100 nm nanoparticles were formed by self-assembly of oppositely charged polyelectrolytes, poly(methacrylic acid) (PMAA) and poly(2-(dimethylamino) ethyl methacrylate) (PDMAEMA) with poly(ethylene glycol) methyl ether methacrylate (PEGMA) acting as a stabilising agent. The encapsulation of hydrophilic and hydrophobic anticancer drugs into the nanoparticle formulations and their release profiles at different pH environments are currently being evaluated.

Introduction and Objectives: Keratoconus is a progressive bilateral degenerative corneal ectasia that affects young adults, causing great visual disability, with a great impact on life quality and social health, worsening over time (1). There is no available medical or pharmacological treatment to prevent the development or decrease its progression. Lactoferrin was observed to promote in vivo and in vitro corneal epithelial wound healing (2). The aim of this work was based on the design, preparation, and characterization of lactoferrin-loaded CS/SBE-b-CD nanoparticles intended for topical ophthalmic administration as a pharmacological alternative for keratoconus treatment.

Methods: Lactoferrin-loaded SBE-β-CD/CS nanoparticles were prepared by ion gelation. Different proportions were tested in order to select the formulation with better characteristics. Evaluation and characterization were carried out in terms of size, surface potential, morphology, production yield (PY), encapsulation efficacy (EE), and loading capacity (LC). Additional experimental data may be included.

Results and discussion: Lactoferrin-loaded SBE-β-CD/CS nanoparticles were prepared, and physicochemical properties successfully evaluated. Proposed nanocarriers show potential as a pharmacological alternative for keratoconus treatment, with the virtue of easy administration, prolonged drug release time, improved ocular bioavailability and reduced dosing frequency.

Conclusions: It is proposed a chitosan-based formulation as a biodegradable mucoadhesive nanocarrier for the controlled delivery of lactoferrin, supported by a preclinical consistent base as the first pharmacological treatment for keratoconus as an alternative to the current invasive clinical methods.

References

[1] Kymes SM, Walline JJ, Zadnik K, Sterling J, Gordon MO, Collaborative Longitudinal Evaluation of Keratoconus Study Group. Changes in the quality-of-life of people with keratoconus. Am J Ophthalmol. 2008 Apr;145(4):611–7.

[2] Pattamatta U, Willcox M, Stapleton F, Cole N, Garrett Q. Bovine lactoferrin stimulates human corneal epithelial alkali wound healing in vitro. Invest Ophthalmol Vis Sci. 2009 Apr;50(4):1636–43.

Triple-negative breast cancer (TNBC) is one of the deadliest types of cancer for women of different age groups. Frequently this cancer does not respond to conservative treatment. Combinatorial RNAi can be suggested as an advanced approach to TNBC therapy. Due to the fact that TNBC cells overexpress chemokine receptor 4, we used modular L1 peptide nanoparticles modified with CXCR4 ligand for combinatorial delivery of siRNAs suppressing major transduction pathways.

TNBC cell line MDA-MB-231 was used as a cellular model. Genes encoding the AQP3, CDC20, and COL4A2 proteins responsible for proliferative activity in TNBC cells were selected as RNAi targets. The siRNA binding ability of the carrier was studied at different charge ratios. The silencing specificity was demonstrated for all siRNAs studied.

AlamarBlue exclusion assay has shown a significant reduction in the anti-proliferative activity after combinatorial siRNA transfection compared to single siRNA delivery. The most significant synergistic effects have been demonstrated after combinatorial transfection with anti-CDC20 siRNA.

Based on our findings, we have concluded that combinatorial treatment by L1-polyplexes formed with AQP3, CDC20, and COL4A2 siRNAs effectively inhibits proliferation of TNBC cells and can be suggested as useful tool for RNAi-mediated cancer therapy.

ABSTRACT:

Polyethyleneimine (PEI) is a synthetic cationic polymer recognized as a non-viral gene carrier with high transfection efficiency [1]. However, cytotoxicity issues limit its use. Pluronic block-copolymers conjugated with PEI have demonstrated promising results for multiparametric target gene/drug co-delivery in cancer with reduced side-effects [1,2].

The goal of this work was to synthesize and characterize a novel nanosystem Pluronic L121-PEI for gene/drug co-delivery.

For this purpose, hydroxyl groups from Pluronic were activated using acryloyl chloride leading to the synthesis of a diacrylate intermediate which was further conjugated with PEI. FTIR and ¹H-NMR spectroscopy were used for structural characterization. Particle size, polydispersity index (PDI) and zeta potential were assessed by Dynamic and Electrophoretic Light Scattering, respectively. A fluorescent pyrene probe was used to evaluate the Critical Micellar Concentration (CMC). Hemolysis experiment was performed to estimate the in vitro biocompatibility of the nanosystem.

FTIR analysis showed that pluronic diacrylate was successfully synthetized as a new band around 1730 cm⁻¹ (C=O bond) appears. Its conjugation with PEI was confirmed by the presence of a band between 3380-3390 cm⁻¹ (N-H bond). ¹H-NMR results showed characteristic proton peaks from Pluronic (-CH₃ at δ1.1 ppm) and from PEI (-CH₂-CH₂NH- between δ2.7–3.4 ppm) and the molar ratio Pluronic-PEI was 1:2. Nanoparticles hydrodynamic diameter was ca. 125 nm with a PDI below 0.250, and a charge nearby +30 mV. The CMC was around 50 μg/mL. The hemolysis ratio of a 5 mg/mL nanomicellar solution was less than 5%.

A novel Pluronic L121-PEI was successfully synthesized which is able to self-assemble in aqueous solution leading to the formation of biocompatible cationic polymeric micelles. Their small size is suitable for tumor-targeting and as they are positively charged they can be also valuable for gene delivery. Overall, this new nanosystem could be a promising multiparametric nanoapproach for gene/drug co-delivery in cancer therapy.

FUNDING:

This work received financial support from National Funds (FCT/MEC, Fundação para a Ciência e Tecnologia/Ministério da Educação e Ciência) through the project UID/QUI/50006/2013, co-financed by European Union (FEDER under the Partnership Agreement PT2020). It was also supported by the grant FCT PTDC/BTM-MAT/30255/2017 (POCI-01- 0145-FEDER-030255) from the Portuguese Foundation for Science and Technology (FCT) and the European Community Fund (FEDER) through the COMPETE2020 program.

REFERENCES:

1. Domingues, C.S. da C.; Serambeque, B.P.; Laranjo Cândido, M.S.; Marto, C.M.M.; Veiga, F.J. de B.; Sarmento Antunes Cruz Ribeiro, A.B.; Figueiras, A.R.R.; Botelho, M.F.R.; Dourado, M. de A.R.F. Epithelial-mesenchymal transition and microRNAs: Challenges and future perspectives in oral cancer. Head Neck 2018, doi:10.1002/hed.25381.
2. Domingues, C.; Alvarez-Lorenzo, C.; Concheiro, A.; Veiga, F.; Figueiras, A. Nanotheranostic Pluronic-Like Polymeric Micelles: Shedding Light into the Dark Shadows of Tumors. Mol. Pharm. 2019, 16, 4757–4774, doi:10.1021/acs.molpharmaceut.9b00945.