Simultaneous Chromatographic Quantitation of Drug Substance and Excipients in Nanoformulations Using a Combination of Evaporative Light Scattering and Absorbance Detectors.

Nanomedicines including lipid- and polymer-based nanoparticles and polymer-drug conjugates enable targeted drug delivery for the treatment of numerous diseases. Quantitative analysis of components in nanomedicines is routinely performed to characterize the products to ensure quality and property consistency but has been mainly focused on the active pharmaceutical ingredients (APIs) in academic publications. It has been increasingly recognized that excipients in nanomedicines are critical in determining the product quality, stability, consistency, and safety. APIs are often analyzed by high-performance liquid chromatography (HPLC), and it would be convenient if the same method can be applied to excipients to robustly quantify all components in nanomedicines. Here, we report the development of a HPLC method that combined an evaporative light scattering (ELS) detector with an UV-vis detector to simultaneously analyze drugs and excipients in nanomedicines. This method was tested on diverse nanodrug delivery systems, including a niosomal nanoparticle encapsulating a phytotherapeutic, a liposome encapsulating an immune boosting agent, and a PEGylated peptide. This method can be utilized for a variety of applications, such as monitoring drug loading, studying drug release, and storage stability. The information obtained from the analyses is of importance for nanomedicine formulation development.

In Vitro Properties and Pharmacokinetics of Temporarily PEGylated Onc72 Prodrugs.

The favorable properties of antimicrobial peptides (AMPs) to rapidly kill pathogens are often limited by unfavorable pharmacokinetics due to fast degradation and renal clearance rates. Here, a prodrug strategy linking proline-rich AMP Onc72 to polyethylene glycol (PEGs) with average molecular weights of 5 and 20 kDa via a peptide linker containing a protease cleavage site is tested for the first time in vivo. Onc72 is released from these 5k- and 20k-prodrugs in mouse serum with half-life times (t1/2 ) of 8 and 14 h, respectively. Importantly, PEGylation protects Onc72 from proteolytic degradation providing a prolonged release of Onc72, balancing the degradation of free Onc72, and leading to relatively stable Onc72 concentrations and high antibacterial activities. The prodrugs are not hemolytic on human erythrocytes and show only slight cytotoxic effects on human cell lines indicating promising safety margins. When administered subcutaneously to female CD-1 mice, the prodrugs elimination t1/2 are 66 min and ≈5.5 h, respectively, compared to 43 min of free Onc72. The maximal Onc72 plasma levels are obtained ≈1 and ≈8 h postadministration, respectively. In conclusion, the prodrugs provide extended elimination t1/2 and a constant release of Onc72 in mice, potentially limiting adverse effects and increasing efficacy.

Antimicrobial Activity and 70S Ribosome Binding of Apidaecin-Derived Api805 with Increased Bacterial Uptake Rate.

In view of the global spread of multiresistant bacteria and the occurrence of panresistant bacteria, there is an urgent need for antimicrobials with novel modes of action. A promising class is antimicrobial peptides (AMPs), including them proline-rich AMPs (PrAMPs), which target the 70S ribosome to inhibit protein translation. Here, we present a new designer peptide, Api805, combining the N- and C-terminal sequences of PrAMPs Api137 and drosocin, respectively. Api805 was similarly active against two Escherichia coli B strains but was inactive against E. coli K12 strain BW25113. These different activities could not be explained by the dissociation constants measured for 70S ribosome preparations from E. coli K12 and B strains. Mutations in the SbmA transporter that PrAMPs use to pass the inner membrane or proteolytic degradation of Api805 by lysate proteases could not explain this either. Interestingly, Api805 seems not to bind to the known binding sites of PrAMPs at the 70S ribosome and inhibited in vitro protein translation, independent of release factors, most likely using a "multimodal effect". Interestingly, Api805 entered the E. coli B strain Rosetta faster and at larger quantities than the E. coli K-12 strain BW25113, which may be related to the different LPS core structure. In conclusion, slight structural changes in PrAMPs significantly altered their binding sites and mechanisms of action, allowing for the design of different antibiotic classes.

Development of novel ultrashort antimicrobial peptide nanoparticles with potent antimicrobial and antibiofilm activities against multidrug-resistant bacteria.

Conventional antibiotics are facing strong microbial resistance that has recently reached critical levels. This situation is leading to significantly reduced therapeutic potential of a huge proportion of antimicrobial agents currently used in clinical settings. Antimicrobial peptides (AMPs) could provide the medical community with an alternative strategy to traditional antibiotics for combating microbial resistance. However, the development of AMPs into clinically useful antibiotics is hampered by their relatively low stability, toxicity, and high manufacturing costs. In this study, a novel in-house-designed potent ultrashort AMP named RBRBR was encapsulated into chitosan-based nanoparticles (CS-NPs) based on the ionotropic gelation method. The encapsulation efficacy reported for RBRBR into CS-NPs was 51.33%, with a loading capacity of 10.17%. The release kinetics of RBRBR from the nanocarrier exhibited slow release followed by progressive linear release for 14 days. The antibacterial kinetics of RBRBR-CS-NPs was tested against four strains of Staphylococcus aureus for 4 days, and the developed RBRBR-CS-NPs exhibited a 3-log decrease in the number of colonies when compared to CS-NP and a 5-log decrease when compared to control bacteria. The encapsulated peptide NP formulation managed to limit the toxicity of the free peptide against both mammalian cells and human erythrocytes. Additionally, the peptide NPs demonstrated up to 98% inhibition of biofilm formation when tested against biofilm-forming bacteria. Loading RBRBR into CS-NPs could represent an innovative approach to develop delivery systems based on NP technology for achieving potent antimicrobial effects against multidrug-resistant and biofilm-forming bacteria, with negligible systemic toxicity and reduced synthetic costs, thereby overcoming the obstructions to clinical development of AMPs.