Polyphosphate coated nanoparticles: Enzyme-activated charge-reversal gene delivery systems.

AIM: The current study aimed to develop enzyme-activated charge-reversal lipid nanoparticles (LNPs) as novel gene delivery systems. METHODS: Palmitic acid was covalently bound to protamine being utilised as transfection promoter to anchor it on the surfaces of LNPs. Green fluorescent protein (GFP) encoding plasmid DNA (pDNA) was ion paired with various cationic counter ions to achieve high encapsulation in LNPs. Protamine-decorated LNPs were prepared by solvent injection method followed by coating with sodium tripolyphosphate (TPP) to generate a bio-inert anionic outer surface. Resulting LNPs were characterised regarding size, polydispersity, zeta potential and encapsulation efficiency. Enzyme-triggered charge-reversal of LNPs was investigated using isolated alkaline phosphatase (ALP) monitoring changes in zeta potential as well as monophosphate release. Furthermore, monophosphate release, cell viability and transfection efficiency were evaluated on a human alveolar epithelial (A549) cell line. RESULTS: Protamine-decorated and TPP-coated (Prot-pDNA/DcChol-TPP) LNPs displayed a mean size of 298.8 ± 17.4 nm and a zeta potential of -13.70 ± 0.61 mV. High pDNA encapsulation was achieved with hydrophobic ion pairs of pDNA with 3ß-[N-(N',N'-dimethylaminoethane)-carbamoyl]cholesterol hydrochloride (DcChol). Zeta potential of Prot-pDNA/DcChol-TPP LNPs reversed to positive values with a total Δ26.8 mV shift upon incubation with ALP. Conformably, a notable amount of monophosphate was released upon incubation of Prot-pDNA/DcChol-TPP LNPs with isolated as well as cell-associated ALP. A549 cells well tolerated LNPs displaying more than 95 % viability. Compared with naked pDNA, unmodified LNPs and control LNPs, Prot-pDNA/DcChol-TPP LNPs showed a significantly increased transfection efficiency. CONCLUSION: Prot-pDNA/DcChol-TPP LNPs can be regarded as promising gene delivery systems.

Zeta potential shifting nanoemulsions comprising single and gemini tyrosine-based surfactants.

AIM: This study aims to design and evaluate zeta potential shifting nanoemulsions comprising single and gemini type tyrosine-based surfactants for specific cleavage by tyrosine phosphatase. METHODS: Tyrosine-based surfactants, either single 4-(2-amino-3-(dodecylamino)-3-oxopropyl)phenyl dihydrogen phosphate (AF1) or gemini 4-(2-amino-3-((1-(dodecylamino)-3-(4-hydroxyphenyl)-1-oxopropan-2-yl)amino)-3-oxopropyl)phenyl dihydrogen phosphate (AF2) type were synthesized via amide bond formation of tyrosine with dodecylamine followed by phosphorylation. These surfactants were incorporated into nanoemulsions. Nanoemulsions were monitored by incubation with isolated tyrosine phosphatase as well as secreted tyrosine phosphatase of Escherichia coli in terms of phosphate release and zeta potential change. RESULTS: Via isolated tyrosine phosphatase, and mediated by E. coli, phosphate groups of either single or gemini tyrosine-based surfactants could be cleaved by secreted tyrosine phosphatase. Nanoemulsions comprising a single tyrosine-based surfactant resulted in a charge shift from - 13.46 mV to - 4.41 mV employing isolated tyrosine phosphatase whilst nanoemulsions consisting of a gemini tyrosine-based surfactant showed a shift in zeta potential from - 15.92 mV to - 5.86 mV, respectively. CONCLUSION: Nanoemulsions containing tyrosine-based surfactants represent promising zeta potential shifting nanocarrier systems targeting tyrosine phosphatase secreting bacteria.

Peptide Antibiotic-Polyphosphate Nanoparticles: A Promising Strategy to Overcome the Enzymatic and Mucus Barrier of the Intestine.

The aim of this study was to develop peptide antibiotic-polyphosphate nanoparticles that are able to overcome the enzymatic and mucus barriers providing a targeted drug release directly on the intestinal epithelium. Polymyxin B-polyphosphate nanoparticles (PMB-PP NPs) were formed via ionic gelation between the cationic peptide and the anionic polyphosphate (PP). The resulting NPs were characterized by particle size, polydispersity index (PDI), zeta potential, and cytotoxicity on Caco-2 cells. The protective effect of these NPs for incorporated PMB was evaluated via enzymatic degradation studies with lipase. Moreover, mucus diffusion of NPs was investigated with porcine intestinal mucus. Isolated intestinal alkaline phosphatase (IAP) was employed to trigger the degradation of NPs and consequent drug release. PMB-PP NPs exhibited an average size of 197.13 ± 14.13 nm, a PDI of 0.36, a zeta potential of -11.1 ± 3.4 mV and a concentration and time-dependent toxicity. They provided entire protection toward enzymatic degradation and exhibited significantly (p < 0.05) higher mucus permeating properties than PMB. When incubated with isolated IAP for 4 h, monophosphate and PMB were constantly released from PMB-PP NPs and zeta potential raised up to -1.9 ± 0.61 mV. According to these findings, PMB-PP NPs are promising delivery systems to protect cationic peptide antibiotics against enzymatic degradation, to overcome the mucus barrier and to provide drug release directly at the epithelium.

Phosphatase-degradable nanoparticles: A game-changing approach for the delivery of antifungal proteins.

HYPOTHESIS: Polyphosphate nanoparticles as phosphatase-degradable carriers for Penicillium chrysogenum antifungal protein (PAF) can enhance the antifungal activity of the protein against Candida albicans biofilm. EXPERIMENTS: PAF-polyphosphate (PP) nanoparticles (PAF-PP NPs) were obtained through ionic gelation. The resulting NPs were characterized in terms of their particle size, size distribution and zeta potential. Cell viability and hemolysis studies were carried out in vitro on human foreskin fibroblasts (Hs 68 cells) and human erythrocytes, respectively. Enzymatic degradation of NPs was investigated by monitoring release of free monophosphates in the presence of isolated as well as C. albicans-derived phosphatases. In parallel, shift in zeta potential of PAF-PP NPs as a response to phosphatase stimuli was determined. Diffusion of PAF and PAF-PP NPs through C. albicans biofilm matrix was analysed by fluorescence correlation spectroscopy (FCS). Antifungal synergy was evaluated on C. albicans biofilm by determining the colony forming units (CFU). FINDINGS: PAF-PP NPs were obtained with a mean size of 300.9 ± 4.6 nm and a zeta potential of -11.2 ± 2.8 mV. In vitro toxicity assessments revealed that PAF-PP NPs were highly tolerable by Hs 68 cells and human erythrocytes similar to PAF. Within 24 h, 21.9 ± 0.4 µM of monophosphate was released upon incubation of PAF-PP NPs having final PAF concentration of 156 µg/ml with isolated phosphatase (2 U/ml) leading to a shift in zeta potential up to -0.7 ± 0.3 mV. This monophosphate release from PAF-PP NPs was also observed in the presence of C. albicans-derived extracellular phosphatases. The diffusivity of PAF-PP NPs within 48 h old C. albicans biofilm matrix was similar to that of PAF. PAF-PP NPs enhanced antifungal activity of PAF against C. albicans biofilm decreasing the survival of the pathogen up to 7-fold in comparison to naked PAF. In conclusion, phosphatase-degradable PAF-PP NPs hold promise as nanocarriers to augment the antifungal activity of PAF and enable its efficient delivery to C. albicans cells for the potential treatment of Candida infections.

Thiolated cyclodextrins: A comparative study of their mucoadhesive properties.

AIM: The aim of this study was the comparison of the mucoadhesive properties of nonionic, negatively, and positively charged thiolated cyclodextrins (CDs), including α-, ß-, and γ-CDs of low and high degree of thiolation. METHODS: Native α-, ß-, and γ-CDs were thiolated with phosphorous pentasulfide in sulfolane (CD-SH) (i), via reductive amination with cysteamine after oxidative ring opening (CD-Cya) (ii), and via esterification with mercaptosuccinic acid (CD-MSA) (iii). These thiolated CDs were characterized via 1H NMR and Ellman's test. Cytotoxicity was determined via resazurin and hemolysis assay. Mucoadhesive properties were evaluated via rheological studies with freshly isolated porcine mucus, as well as residence time studies on porcine small intestinal mucosa. RESULTS: The structure of thiolated CDs was confirmed via 1H NMR. The degree of thiolation was in the range of 594-1034 µmol/g for low and 1360-3379 µmol/g for high CD-SH, whereas thiolated CD-Cya and thiolated CD-MSA exhibited a degree of thiolation of 1142-3242 µmol/g and 243-1227 µmol/g, respectively. Just cationic CDs showed cytotoxicity. Nonionic highly thiolated α-CD-SH, α-CD-Cya, and α-CD-MSA exhibited with mucus 5.6-, 15.7- and 2.8-fold improved dynamic viscosity, while improvement was 7.7-, 6.1-, and 5.4-fold for the corresponding thiolated ß-CDs and 12.3-, 15.4- and 17.8-fold for the corresponding thiolated γ-CDs compared with native CDs, respectively. A prolonged mucosal residence time following the rank order γ > ß > α was observed for all thiolated CDs, whereby γ-CD-Cya, nonionic highly thiolated ß-CD-SH and α-CD-Cya showed the highest mucoadhesive properties. CONCLUSION: A high degree of thiolation and the introduction of cationic charges are mainly responsible for high mucoadhesive properties of CDs.

Nanoarchitectonics of Layer-by-Layer (LbL) coated nanostructured lipid carriers (NLCs) for Enzyme-Triggered charge reversal.

HYPOTHESIS: Combined usage of Layer-by-Layer (LbL) coating and alkaline phosphatase (ALP) - responsive charge reversal strategies can improve the cellular internalisation of the colloidal drug delivery systems by also decreasing their cytotoxic effects. EXPERIMENTS: Anionic core NLCs were formed by combining the melt emulsification method and ultrasonication. The resulting core NLCs were coated sequentially first with protamine (Prot NLCs) and then with sodium tripolyphosphate (TPP) or sodium polyphosphate (Graham's salt, PP) generating TPP or PP NLCs, respectively. The developed NLCs were characterised regarding their size and zeta potential. Enzyme-induced charge reversal of the TPP and PP NLCs was evaluated by zeta potential measurements upon their incubation with alkaline phosphatase (ALP). In parallel, time-dependent phosphate release was monitored in the presence of isolated as well as cell-associated ALP. Morphological evaluations were performed by scanning electron microscopy (SEM) studies. Moreover, cell viability and cellular uptake studies were carried out in vitro on Caco-2 cells. FINDINGS: The core NLCs were obtained with a mean size of 272.27 ± 5.23 nm and a zeta potential of -25.70 ± 0.26 mV. Upon coating with protamine, the zeta potential raised to positive values with a total change up to Δ29.3 mV also displaying an increase in particle size. The second layer coating with TPP and PP provided a negative surface charge. Subsequent to ALP treatment, the zeta potential of the TPP and PP NLCs reversed from negative to positive values with total changes of Δ8.56 and Δ7.47 mV, respectively. Conformably, significant amounts of phosphate were released from both formulations. Compared with core NLCs, improved cellular viability as well as increased cellular uptake were observed in case of Prot, TPP and PP NLCs.

Chitosan - Polyphosphate nanoparticles for a targeted drug release at the absorption membrane.

The aim of this study was to develop nanoparticles (NPs) providing a targeted drug release directly on the epithelium of the intestinal mucosa. NPs were prepared via ionic gelation between cationic chitosan (Cs) and anionic polyphosphate (PP). The resulting NPs were characterized by their size, polydispersity index (PDI) and zeta potential. Isolated and cell-associated intestinal alkaline phosphatase (IAP) was employed to trigger polyphosphate cleavage in Cs-PP NPs which was quantified via malachite green assay. In parallel, the shift in zeta potential was determined. In-vitro drug release studies were performed in Franz diffusion cells with Cs-PP NPs containing rhodamine 123 as model active ingredient. Furthermore, cytotoxicity of Cs-PP NPs was assessed via resazurin assay on Caco-2 cells as well as via hemolysis assay on red blood cells. Cs-PP NPs exhibited an average size of 144.17 ± 10.95 nm and zeta potential of -12.6 ± 0.50 mV. The encapsulation efficiency of rhodamine 123 by Cs-PP NPs was 86.8%. After incubation with isolated IAP for 3 h the polyphosphate of Cs-PP NPs was cleaved to monophosphate and zeta potential raised up to -2.3 ± 0.30 mV. Cs-PP NPs showed a non-toxic profile. Within 3 h, 62.0 ± 10.8% and 14.1 ± 2.2% of total rhodamine 123 was released from Cs-PP NPs upon incubation with isolated as well as porcine intestine derived intestinal alkaline phosphatase (IAP), respectively. According to these results, Cs-PP NPs are promising drug delivery systems to enable a drug targeted release at the absorption membrane.

Design of nanostructured lipid carriers and solid lipid nanoparticles for enhanced cellular uptake.

In this study PEG-free and zeta potential changing lipid-based nanocarriers providing enhanced cellular uptake were developed. Nanostructured lipid carriers (NLC), consisting of paraffin wax, caprylic/ capric triglyceride, cetyltrimethylammoniumchloride and either soy lecithin or polyglycerol-4 laurate and solid lipid nanoparticles (SLN) with the same composition but without the liquid lipid content were developed. All formulations exposed a positive surface charge and were then coated with the polyphosphate Graham's salt. Phosphate release from these formulations was evaluated by incubation with intestinal alkaline phosphatase as well as on a Caco-2 monolayer and zeta potentials were measured. Additionally, cellular uptake studies were performed. Within 5 h, a remarkable amount of phosphate was released from all formulations incubated with intestinal alkaline phosphatase. Enzymatically induced phosphate release with intestinal alkaline phosphatase led to a zeta potential shift up to Δ 26 mV. Results of phosphate release and zeta potential change were confirmed on Caco-2 cells. Cellular uptake studies on Caco-2 cells showed an up to 5.6-times higher uptake compared to cells with inhibited phosphatase. According to these results, polyphosphate coating is a powerful tool to obtain lipid-based nanocarriers for enhanced cellular uptake.

Charge reversal self-emulsifying drug delivery systems: A comparative study among various phosphorylated surfactants.

HYPOTHESIS: Phosphorylated surfactants having ethoxylate spacer arms are promising excipients for charge reversal self-emulsifying drug delivery systems (SEDDS). EXPERIMENTS: 1,2-Dipalmitoyl-sn-glycero-3-phosphatidic acid disodium salt (PA), 2-((2,3-bis(oleoyloxy)propyl)dimethylammonio)ethyl hydrogen phosphate (DOCP), nonylphenol monophosphate ester (PNPP), C12-15 alcohol 3 ethoxylate phosphate ester (PME) and polyoxyethylene (9) dioctanoyl glycerol pyrophosphate (DGPP) loaded SEDDS were developed and characterized. Zeta potential of SEDDS was measured before and after incubation with intestinal alkaline phosphatase (IAP). Phosphate release was monitored by incubation of SEDDS with isolated as well as cell-associated IAP. Primary amine content on the surface of SEDDS was determined in parallel. Cytotoxicity was evaluated on Caco-2 cells and in vitro hemolysis test was performed. Cellular uptake studies were performed by confocal scanning microscopy. FINDINGS: SEDDS formulations exhibited a size in the range of 17 and 193 nm and a polydispersity index (PDI) ≤ 0.5. Charge reversal from negative to positive values could be achieved in case of PNPP and PME loaded SEDDS with a zeta potential changing from -13 mV to +9 mV and from -7 to +2 mV, respectively, within 6 h. Significant amounts of phosphate were released from PNPP and PME loaded SEDDS incubated with isolated IAP and from all formulations incubated with cell-associated IAP in accordance with an increase in primary amines on the surface of oily droplets. SEDDS exhibited a concentration and time-dependent cytotoxicity. PNPP and PME SEDDS displayed an increased cellular uptake.

Less Reactive Thiol Ligands: Key towards Highly Mucoadhesive Drug Delivery Systems.

As less reactive s-protected thiomers can likely interpenetrate the mucus gel layer to a higher extent before getting immobilized via disulfide bond formation with mucins, it was the aim of this study to develop a novel type of s-protected thiomer based on the less reactive substructure cysteine-N-acetyl cysteine (Cys-NAC) in order to obtain improved mucoadhesive properties. For this purpose, two types of s-protected thiomers, polyacrylic acid-cysteine-mercaptonicotinic acid (PAA-Cys-MNA) and polyacrylic acid-cysteine-N-acetyl cysteine (PAA-Cys-NAC), were synthesized and characterized by Fourier-transform infrared spectroscopy (FT-IR) and the quantification of attached disulfide ligands. The viscosity of both products was measured in the presence of NAC and mucus. Both thiomers were also evaluated regarding swelling behavior, tensile studies and retention time on the porcine intestinal mucosa. The FT-IR spectra confirmed the successful attachment of Cys-MNA and Cys-NAC ligands to PAA. The number of attached sulfhydryl groups was in the range of 660-683 µmol/g. The viscosity of both s-protected thiomers increased due to the addition of increasing amounts of NAC. The viscosity of the mucus increased in the presence of 1% PAA-Cys-MNA and PAA-Cys-NAC 5.6- and 10.9-fold, respectively, in comparison to only 1% PAA. Both s-protected thiomers showed higher water uptake than unmodified PAA. The maximum detachment force (MDF) and the total work of adhesion (TWA) increased in the case of PAA-Cys-MNA up to 1.4- and 1.6-fold and up to 2.4- and 2.8-fold in the case of PAA-Cys-NAC. The retention of PAA, PAA-Cys-MNA, and PAA-Cys-NAC on porcine intestinal mucosa was 25%, 49%, and 76% within 3 h, respectively. The results of this study provide evidence that less reactive s-protected thiomers exhibit higher mucoadhesive properties than highly reactive s-protected thiomers.