Cyanocobalamin-Modified Colistin-Hyaluronan Conjugates: Synthesis and Bioactivity.

Polymeric drug delivery systems enhance the biopharmaceutical properties of antibiotics by increasing their bioavailability, providing programmable and controlled-release properties, and reducing toxicity. In addition, drug delivery systems are a promising strategy to improve the intestinal permeability of various antimicrobial agents, including colistin (CT). This study describes the modification of conjugates based on CT and hyaluronic acid (HA) with cyanocobalamin (vitamin B12). Vitamin B12 was chosen as a targeting ligand because it has its own absorption pathway in the small intestine. The resulting polysaccharide conjugates contained 95 µg/mg vitamin B12 and the CT content was 335 µg/mg; they consisted of particles of two sizes, 98 and 702 nm, with a ζ-potential of approximately -25 mV. An in vitro release test at pH 7.4 and pH 5.2 showed an ultra-slow release of colistin of approximately 1% after 10 h. The modified B12 conjugates retained their antimicrobial activity at the level of pure CT (minimum inhibitory concentration was 2 µg/mL). The resulting delivery systems also reduced the nephrotoxicity of CT by 30-40% (HEK 293 cell line). In addition, the modification of B12 improved the intestinal permeability of CT, and the apparent permeability coefficient of HA-CT-B12 conjugates was 3.5 × 10-6 cm/s, corresponding to an in vivo intestinal absorption of 50-100%. Thus, vitamin-B12-modified conjugates based on CT and HA may be promising oral delivery systems with improved biopharmaceutical properties.

Succinyl Chitosan-Colistin Conjugates as Promising Drug Delivery Systems.

The growth of microbial multidrug resistance is a problem in modern clinical medicine. Chemical modification of active pharmaceutical ingredients is an attractive strategy to improve their biopharmaceutical properties by increasing bioavailability and reducing drug toxicity. Conjugation of antimicrobial drugs with natural polysaccharides provides high efficiency of these systems due to targeted delivery, controlled drug release and reduced toxicity. This paper reports a two-step synthesis of colistin conjugates (CT) with succinyl chitosan (SucCS); first, we modified chitosan with succinyl anhydride to introduce a carboxyl function into the polymer molecule, which was then used for chemical grafting with amino groups of the peptide antibiotic CT using carbodiimide chemistry. The resulting polymeric delivery systems had a degree of substitution (DS) by CT of 3-8%, with conjugation efficiencies ranging from 54 to 100% and CT contents ranging from 130-318 µg/mg. The size of the obtained particles was 100-200 nm, and the ζ-potential varied from -22 to -28 mV. In vitro release studies at pH 7.4 demonstrated ultra-slow hydrolysis of amide bonds, with a CT release of 0.1-0.5% after 12 h; at pH 5.2, the hydrolysis rate slightly increased; however, it remained extremely low (1.5% of CT was released after 12 h). The antimicrobial activity of the conjugates depended on the DS. At DS 8%, the minimum inhibitory concentration (MIC) of the conjugate was equal to the MIC of native CT (1 µg/mL); at DS of 3 and 5%, the MIC increased 8-fold. In addition, the developed systems reduced CT nephrotoxicity by 20-60%; they also demonstrated the ability to reduce bacterial lipopolysaccharide-induced inflammation in vitro. Thus, these promising CT-SucCS conjugates are prospective for developing safe and effective nanoantibiotics.

Synthesis and Characterization of Novel Succinyl Chitosan-Dexamethasone Conjugates for Potential Intravitreal Dexamethasone Delivery.

The development of intravitreal glucocorticoid delivery systems is a current global challenge for the treatment of inflammatory diseases of the posterior segment of the eye. The main advantages of these systems are that they can overcome anatomical and physiological ophthalmic barriers and increase local bioavailability while prolonging and controlling drug release over several months to improve the safety and effectiveness of glucocorticoid therapy. One approach to the development of optimal delivery systems for intravitreal injections is the conjugation of low-molecular-weight drugs with natural polymers to prevent their rapid elimination and provide targeted and controlled release. This study focuses on the development of a procedure for a two-step synthesis of dexamethasone (DEX) conjugates based on the natural polysaccharide chitosan (CS). We first used carbodiimide chemistry to conjugate DEX to CS via a succinyl linker, and we then modified the obtained systems with succinic anhydride to impart a negative ζ-potential to the polymer particle surface. The resulting polysaccharide carriers had a degree of substitution with DEX moieties of 2-4%, a DEX content of 50-85 µg/mg, and a degree of succinylation of 64-68%. The size of the obtained particles was 400-1100 nm, and the ζ-potential was -30 to -33 mV. In vitro release studies at pH 7.4 showed slow hydrolysis of the amide and ester bonds in the synthesized systems, with a total release of 8-10% for both DEX and succinyl dexamethasone (SucDEX) after 1 month. The developed conjugates showed a significant anti-inflammatory effect in TNFα-induced and LPS-induced inflammation models, suppressing CD54 expression in THP-1 cells by 2- and 4-fold, respectively. Thus, these novel succinyl chitosan-dexamethasone (SucCS-DEX) conjugates are promising ophthalmic carriers for intravitreal delivery.

Hyaluronan/Diethylaminoethyl Chitosan Polyelectrolyte Complexes as Carriers for Improved Colistin Delivery.

Improving the therapeutic characteristics of antibiotics is an effective strategy for controlling the growth of multidrug-resistant Gram-negative microorganisms. The purpose of this study was to develop a colistin (CT) delivery system based on hyaluronic acid (HA) and the water-soluble cationic chitosan derivative, diethylaminoethyl chitosan (DEAECS). The CT delivery system was a polyelectrolyte complex (PEC) obtained by interpolymeric interactions between the HA polyanion and the DEAECS polycation, with simultaneous inclusion of positively charged CT molecules into the resulting complex. The developed PEC had a hydrodynamic diameter of 210-250 nm and a negative surface charge (ζ-potential = -19 mV); the encapsulation and loading efficiencies were 100 and 16.7%, respectively. The developed CT delivery systems were characterized by modified release (30-40% and 85-90% of CT released in 15 and 60 min, respectively) compared to pure CT (100% CT released in 15 min). In vitro experiments showed that the encapsulation of CT in polysaccharide carriers did not reduce its antimicrobial activity, as the minimum inhibitory concentrations against Pseudomonas aeruginosa of both encapsulated CT and pure CT were 1 µg/mL.

Antimicrobial Peptide Arenicin-1 Derivative Ar-1-(C/A) as Complement System Modulator.

Antimicrobial peptides (AMPs) are not only cytotoxic towards host pathogens or cancer cells but also are able to act as immunomodulators. It was shown that some human and non-human AMPs can interact with complement proteins and thereby modulate complement activity. Thus, AMPs could be considered as the base for complement-targeted therapeutics development. Arenicins from the sea polychaete Arenicola marina, the classical example of peptides with a ß-hairpin structure stabilized by a disulfide bond, were shown earlier to be among the most prospective regulators. Here, we investigate the link between arenicins' structure and their antimicrobial, hemolytic and complement-modulating activities using the derivative Ar-1-(C/A) without a disulfide bond. Despite the absence of this bond, the peptide retains all important functional activities and also appears less hemolytic in comparison with the natural forms. These findings could help to investigate new complement drugs for regulation using arenicin derivatives.

Towards lead compounds as anti-cancer agents via new phaeosphaeride A derivatives.

New derivatives of phaeosphaeride A (PPA) were synthesized and characterized. Anti-tumor studies were carried out on the U937, HCT-116, PC3, MCF-7, A549, К562, NCI-H929, Jurkat, THP-1, RPMI8228 tumor cell lines, and on the HEF cell line. All the compounds synthesized were found to have better efficacy than PPA towards the tumor cell lines mentioned. Compound 6 (IC50 = 0.59 ±â€¯0.27 µM) was observed to be 11 times more active than PPA (IC50 = 6.5 ±â€¯0.30 µM) towards the NCI-H929 cell line, with a therapeutic index of 18. Compound 6 was determined to be over half and 16 times more active than etoposide towards the NCI-H929 (IC50 = 0.9 ±â€¯0.05 µM) and A549 (IC50 = 100 ±â€¯7.0 µM) cell lines, respectively.

Hyaluronan/B12-chitosan polyelectrolyte complex for oral colistin administration.

Polyelectrolyte complexes (PECs) based on polysaccharides, including hyaluronic acid (HA) and chitosan (CS), are promising delivery systems for antimicrobial agents, including oral administration of the peptide antibiotic colistin (CT). Modification of CS with different targeting ligands to improve intestinal permeability is a suitable way to improve the oral bioavailability of polyelectrolyte particles. This study describes the procedure for obtaining CT-containing PECs based on HA and CS modified with cyanocobalamin (vitamin B12). In this case, vitamin B12 is used as a targeting ligand because it is absorbed in the ileum via specific transporter proteins. The resulting PECs had a hydrodynamic size of about 284 nm and a positive ζ-potential of about 26 mV; the encapsulation efficiency was 88.2 % and the CT content was 42.2 µg/mg. The developed systems provided a two-phase drug release: about 50 % of the CT was released in 0.5-1 h, and about 60 % of the antibiotic was cumulatively released in 5 h. The antimicrobial activity of encapsulated CT was maintained at the same level as the pure drug for at least 24 h (minimum inhibitory concentration against Pseudomonas aeruginosa was 2 µg/mL for both). In addition, the apparent permeability coefficient of CT in the PEC formulation was 2.4 × 10-6 cm/s. Thus, the incorporation of CT into HA- and vitamin B12-modified CS-based PECs can be considered as a simple and convenient method to improve the oral delivery of CT.

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry of titanium oxide-enriched peptides for detection of aged organophosphorus adducts on human butyrylcholinesterase.

Exposure to nerve agents or organophosphorus (OP) pesticides can have life-threatening effects. Human plasma butyrylcholinesterase (BChE) inactivates these poisons by binding them to Ser198. After hours or days, these OP adducts acquire a negative charge by dealkylation in a process called aging. Our goal was to develop a method for enriching the aged adduct to facilitate detection of exposure. Human BChE inhibited by OP toxicants was incubated for 4 days to 6 years. Peptides produced by digestion with pepsin were enriched by binding to titanium oxide (TiO2) and analyzed by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry. It was found that with two exceptions, all aged OP adducts in peptide FGES198AGAAS were enriched by binding to Titansphere tips. Cresyl saligenin phosphate yielded two types of aged adduct, cresylphosphate and phosphate, but only the phosphate adduct bound to Titansphere. The nerve agent VR yielded no aged adduct, supporting crystal structure findings that the VR adduct on BChE does not age. The irreversible nature of aged OP adducts was demonstrated by the finding that after 6 years at room temperature in sterile pH 7.0 buffer, the adducts were still detectable. It was concluded that TiO2 microcolumns can be used to enrich aged OP-modified BChE peptide.

PHOS-select iron affinity beads enrich peptides for the detection of organophosphorus adducts on albumin.

Albumin is covalently modified by organophosphorus toxicants (OP) on tyrosine 411, but less than 1% of albumin is modified in humans by lethal OP doses that inhibit 95% of plasma butyrylcholinesterase. A method that enriches OP-modified albumin peptides could aid analysis of low dose exposures. Soman or chlorpyrifos oxon treated human plasma was digested with pepsin. Albumin peptides were enriched by binding to Fe(3+) beads at pH 11 and eluted with pH 2.6 buffer. Similarly, mouse and guinea pig albumin modified by chlorpyrifos oxon were digested with pepsin and enriched by binding to Fe(3+) beads. Peptides were identified by MALDI-TOF/TOF mass spectrometry. PHOS-select iron affinity beads specifically enriched albumin peptides VRY411TKKVPQVST and LVRY411TKKVPQVST in a pepsin digest of human plasma. The unmodified as well as OP-modified peptides bound to the beads. The binding capacity of 500 µL of beads was the pepsin digest of 2.1 µL of human plasma. The limit of detection was 0.2% of OP-modified albumin peptide in 0.43 µL of plasma. Enrichment of OP-modified albumin peptides by binding to Fe(3+) beads is a method with potential application to diagnosis of OP pesticide and nerve agent exposure in humans, mice, and guinea pigs.

Hyaluronan-colistin conjugates: Synthesis, characterization, and prospects for medical applications.

The development of nanotechnology-based antibiotic delivery systems (nanoantibiotics) is an important challenge in the effort to combat microbial multidrug resistance. These systems have improved biopharmaceutical characteristics by increasing local bioavailability and reducing systemic toxicity and the number and frequency of drug side effects. Conjugation of low -molecular -weight antibacterial agents with natural polysaccharides is an effective strategy for developing optimal targeted delivery systems with programmed release and reduced cytotoxicity. This study describes the synthesis of conjugates of colistin (CT) and hyaluronic acid (HA) using carbodiimide chemistry to conjugate the amino groups of CT with the carboxyl groups of HA. The obtained polysaccharide carriers had a degree of substitution (DS) with CT molecules of 3-10 %, and the CT content was 129-377 µg/mg. The size of the fabricated particles was 300-600 nm; in addition, there were conjugates in the form of single macromolecules (30-50 nm). The ζ-potential of developed systems was about -20 mV. In vitro release studies at pH 7.4 and pH 5.2 showed slow hydrolysis of amide bonds, with a CT release of 1-5 % after 24 h. The conjugates retained antimicrobial activity depending on the DS: at DS 8 %, the minimum inhibitory concentration (MIC) of the conjugate corresponded to the MIC of free CT. The resulting systems also reduced CT nephrotoxicity by 20-50 %. These new conjugates of CT with HA are promising for the development of nanodrugs for safe and effective antimicrobial therapy.

Hyaluronan/colistin polyelectrolyte complexes: Promising antiinfective drug delivery systems.

Nanotechnology-based modification of known antimicrobial agents is a rational and straightforward way to improve their safety and effectiveness. The aim of this study was to develop colistin (CT)-loaded polymeric carriers based on hyaluronic acid (HA) for potential application as antimicrobial agents against multi-resistant gram-negative microorganisms (including ESKAPE pathogens). CT-containing particles were obtained via a polyelectrolyte interaction between protonated CT amino groups and HA carboxyl groups (the CT-HA complex formation constant [logKCT-HA] was about 5.0). The resulting polyelectrolyte complexes had a size of 210-250 nm and a negative charge (ζ-potential -19 mV), with encapsulation and loading efficiencies of 100% and 20%, respectively. The developed CT delivery systems were characterized by modified release (45% and 85% of CT released in 15 and 60 min, respectively) compared to pure CT (100% CT released in 15 min). In vitro tests showed that the encapsulation of CT in polymer particles did not reduce its pharmacological activity; the minimum inhibitory concentrations of both encapsulated CT and pure CT were 1 µg/mL (against Pseudomonas aeruginosa).