Temperature-sensitive liposomal ciprofloxacin for the treatment of biofilm on infected metal implants using alternating magnetic fields.

Implants are commonly used as a replacement for damaged tissue. Many implants, such as pacemakers, chronic electrode implants, bone screws, and prosthetic joints, are made of or contain metal. Infections are one of the difficult to treat complications associated with metal implants due to the formation of biofilm, a thick aggregate of extracellular polymeric substances (EPS) produced by the bacteria. In this study, we treated a metal prosthesis infection model using a combination of ciprofloxacin-loaded temperature-sensitive liposomes (TSL) and alternating magnetic fields (AMF). AMF heating is used to disrupt the biofilm and release the ciprofloxacin-loaded TSL. The three main objectives of this study were to (1) investigate low- and high-temperature-sensitive liposomes (LTSLs and HTSLs) containing the antimicrobial agent ciprofloxacin for temperature-mediated antibiotic release, (2) characterise in vitro ciprofloxacin release and stability and (3) study the efficacy of combining liposomal ciprofloxacin with AMF against Pseudomonas aeruginosa biofilms grown on metal washers. The release of ciprofloxacin from LTSL and HTSL was assessed in physiological buffers. Results demonstrated a lower transition temperature for both LTSL and HTSL formulations when incubated in serum as compared with PBS, with a more pronounced impact on the HTSLs. Upon combining AMF with temperature-sensitive liposomal ciprofloxacin, a 3 log reduction in CFU of Pseudomonas aeruginosa in biofilm was observed. Our initial studies suggest that AMF exposure on metal implants can trigger release of antibiotic from temperature sensitive liposomes for a potent bactericidal effect on biofilm.

Elastin-like polypeptide incorporated thermally sensitive liposome improve antibiotic therapy against musculoskeletal bacterial pathogens.

Musculoskeletal infections caused by bacteria such as Staphylococcus aureus and Pseudomonas aeruginosa in children and adults can lead to adverse outcomes including a need for extensive surgical debridement and limb amputation. To enable targeted antimicrobial release in infected tissues, the objective of this study was to design and investigate novel elastin-like polypeptide (ELP)-based thermally sensitive liposomes in vitro. ELP biopolymers can change their phase behaviour at higher temperatures. We hypothesised that ELP-TSL will improve therapeutic efficacy by releasing antimicrobial payloads locally at higher temperatures (≥39 °C). ELP-TSL library were formulated by varying cholesterol and phospholipid composition by the thin film and extrusion method. A broad-spectrum antimicrobial (Ciprofloxacin or Cipro) was encapsulated inside the liposomes by the ammonium sulphate gradient method. Cipro release from ELP-TSLs was assessed in physiological buffers containing ∼25% serum by fluorescence spectroscopy, and efficacy against Staphylococcus aureus and Pseudomonas aeruginosa was assessed by disc diffusion and planktonic assay. Active loading of Cipro achieved an encapsulation efficiency of 40-70% in the ELP-TSL depending upon composition. ELP-TSL Cipro release was near complete at ≥39 °C; however, the release rates could be delayed by cholesterol. Triggered release of Cipro from ELP-TSL at ∼42 °C induced significant killing of S. aureus and P. aeruginosa compared to 37 °C. Our in vitro data suggest that ELP-TSL may potentially improve bacterial wound therapy in patients.

Identification of Excipients for Stabilizing Fiberless Adenovirus as Biopharmaceuticals.

Reducing the promiscuous tropism of native adenovirus by using fiberless adenovirus is advantageous toward its use as a gene therapy vector or vaccine component. The removal of the fiber protein on native adenovirus abrogates several undesirable interactions; however, this approach decreases the particle's physical stability. To create stable fiberless adenovirus for pharmaceutical use, the effects of temperature and pH on the particle's stability profile must be addressed. Our results indicate that the stability of fiberless adenovirus is increased when it is stored in mildly acidic conditions around pH 6. The stability of fiberless adenovirus can be further enhanced by using excipients. Excipient screening results indicate that the nonionic surfactant Pluronic F-68 and the amino acid glycine are potential stabilizers because of their ability to increase the thermal transition temperature of the virus particle and promote retention of biological activity after exposure to prolonged thermal stress. Our data indicate that the instability of fiberless adenovirus can be ameliorated by storing the virus in the appropriate environment, and it should be possible to further optimize the virus so that it can be used as a biopharmaceutical.

Effects of cell-penetrating peptides on transduction efficiency of PEGylated adenovirus.

BACKGROUND: Adenovirus (Ad) is one of the viral vectors most widely used for gene delivery. The virus, however, has serious shortcomings such as immunogenicity, promiscuous tropism, and the inability to efficiently infect certain types of cells. The goal of this study was to improve the ability of an Ad-based vector to efficiently transform cells that lack the native coxsackie-adenovirus receptor (CAR(-)) by modifying the virus with CPP-PEG conjugates. METHODS: The vector was produced by PEGylating Ad, which packages a lacZ reporter gene, and then conjugating CPPs to form CPP-PEG-Ad particles. The study compared the effectiveness of four different CPPs: Pen, Tat, Pep1, and pArg. The effects of CPP amount per virus, degree of PEGylation, and PEG molecular weight on transduction efficiency were studied on CAR(-) NIH/3T3 cells. RESULTS: CPP-PEG-Ad particles transduced CAR(-) cells significantly better than unmodified Ad. Pen, the most effective CPP, produced an 80-fold improvement in transduction compared to the unmodified virus. The Pen peptide utilized a combination of electrostatic and hydrophobic interactions with the cell membrane to maximize cellular association while the other CPPs used only electrostatic or hydrophobic interactions but not both. Lastly, higher degrees of PEGylation, which prompted PEG to adopt a "brush" conformation, resulted in more efficient CPP-PEG-Ad particles because of both better conjugation of CPPs to the PEGylated virus and better exposure of the conjugated CPPs on the surface of the particle. CONCLUSIONS: CPP-PEG-Ad particles efficiently deliver genes to cells that Ad alone would not efficiently infect, thereby extending potential gene therapy treatments to a much broader range of cell types and diseases.

Evaluation of cell-penetrating peptide/adenovirus particles for transduction of CAR-negative cells.

Adenovirus (Ad) is a promising gene therapy vector, and is used currently in more than 23% of clinical gene therapy trials. The viral vector, however, has drawbacks such as immunogenicity, promiscuous tropism, and the inability to infect certain types of cells. The focus of this work was to develop an improved vector through electrostatic formation of a complex between negatively charged Ad and positively charged cell-penetrating peptides (CPPs), including Tat, Penetratin, polyarginine, and Pep1. The resulting complexes were demonstrated to be capable of transducing cells that lack the coxsackie-adenovirus receptor (CAR), and are otherwise difficult to infect with native Ad. The transduction efficiency of the complexes was optimized by varying the multiplicity of infection, complex formation time, and ratio of CPPs to Ad, which improved the transduction efficiency of CPP/Ad on CAR-negative cells more than 100-fold compared with unmodified Ad. The size of the CPP/Ad complex was initially less than 300 nm, but stability studies performed in the presence of serum indicate that the complex aggregates with serum after an extended period of time. The results of the current study indicate that electrostatic modification of Ad with CPPs provides a relevant platform for developing effective Ad-based gene therapy vectors.