Anders Elias Hansen

Background: Antibiotic-loaded ceramic bone grafts (ACBGs) are widely assumed to provide extended local antimicrobial protection, maintaining concentrations above the minimum inhibitory concentration (MIC) for 28 to 42 days. However, this time frame is mainly based on in vitro elution assays with simplified washout models, and its relevance to in vivo performance is uncertain. This study compared the in vivo and in vitro release and behavior of gentamicin from 2 commercial ACBGs. Methods: Gentamicin-loaded hydroxyapatite/calcium sulfate (HA-CS) and calcium sulfate (CS) composites were investigated using a murine implantation model and in vitro elution assays. Gentamicin content in recovered depot material and elution media was quantified by liquid chromatography with tandem mass spectrometry. Model calculations evaluated how the assay design affects the estimated time above the MIC. Results: Both ACBGs released >98% of gentamicin within 6 hours and >99% within 24 hours in vivo. In vitro assays likewise indicated early depot depletion, with concentration-time profiles beyond 48 hours driven by serial dilution rather than release. Model calculations suggested that the apparent prolonged antibiotic activity in previous in vitro assays was largely due to the assay design, not to sustained release, implying that freely administered antibiotics could produce similar profiles. Conclusions: Neither ACBG demonstrated measurable sustained release beyond an initial 24-hour burst phase. This may have important clinical implications, as expectations of prolonged local antimicrobial protection are not supported by the cumulative-release data showing rapid antibiotic depot depletion.

The therapeutic challenges of orthopedic device-related infections and emerging antimicrobial resistance have attracted attention to drug delivery technologies. This study evaluates the preclinical efficacy of local single- and dual-antibiotic therapy against implant-associated osteomyelitis (IAO) using a drug-eluting depot technology, CarboCell, that provides sustained release of high-dose antibiotics and allows for strategic in situ placement in relation to infectious lesions. Clindamycin and gentamicin were formulated in CarboCell compositions. One-stage-revision of tibial Staphylococcus aureus IAO was conducted in 19 pigs. Pigs were treated locally with CarboCell containing either gentamicin alone for 1 week or a co-formulation of gentamicin and clindamycin for 1 or 3 weeks. Bone, soft tissue, and antibiotic depots were collected for microbiology, histology, and HPLC analyses. Supporting in vivo release studies of CarboCell formulations were performed on mice. Both single- and dual-antibiotic CarboCell formulations were developed and capable of eradicating the infectious bacteria in bone and preventing colonization of implants inserted at revision. Eradication in soft tissue was observed in all pigs after 3 weeks and in 6/9 pigs after 1 week of treatment. Neutrophil counts in bone tissue were below the infection cut-off in all pigs receiving the dual-antibiotic therapies, but above in all pigs receiving the single-antibiotic therapy. Histological signs of active bone reorganization and healing were observed at 3 weeks. In conclusion, all CarboCell formulations demonstrated strong therapeutic activity against IAO, eradicating S. aureus in bone tissue and preventing colonization of implants even without the addition of systemic antibiotic therapy.

Bone infections from Staphylococcus aureus are notoriously difficult to treat and have high recurrence rates. Local antibiotic delivery systems hold the potential to achieve high in situ antibiotic concentrations, which are otherwise challenging to achieve via systemic administration. Existing solutions have been shown to confer suboptimal drug release and distribution. Here we present and evaluate an injectable in situ-forming depot system termed CarboCell. The CarboCell technology provides sustained and tuneable release of local high-dose antibiotics. CarboCell formulations of levofloxacin or clindamycin with or without antimicrobial adjuvants cis-2-decenoic acid or cis-11-methyl-2-dodecenoic acid were tested in experimental rodent and porcine implant-associated osteomyelitis models. In the porcine models, debridement and treatment with CarboCell-formulated antibiotics was carried out without systemic antibiotic administration. The bacterial burden was determined by quantitative bacteriology. CarboCell formulations eliminated S. aureus in infected implant rat models. In the translational implant-associated pig model, surgical debridement, and injection of clindamycin-releasing CarboCell formulations resulted in pathogen-free bone tissues and implants in 9/12, and full eradication in 5/12 pigs. Sustained release of antimicrobial agents mediated by the CarboCell technology demonstrated promising therapeutic efficacy in challenging translational models and may be beneficial in combination with the current standard of care.

TLR 7/8 agonists are highly potent immunostimulators, though their clinical translation has been met with mixed success, due to their high toxicity as a result of an unregulated systemic immune activation. There is enormous potential to augment cancer immunotherapies with synthetic TLR 7/8 agonists, though a thorough control of pharmacokinetics and localization is needed for the general use of TLR 7/8 agonists in cancer immunotherapy. Herein, we control localization of TLR 7/8 agonists, by exploiting the extensive tissue retention of poly(acrylic acid-co-styrene). In a murine CT26 model, we find that covalently attaching TLR 7/8 agonists to the copolymer allows for retaining the drug in the tumor microenvironment for at least 15 weeks, after intratumoral injection, and results in a curative monotherapy. The copolymer itself is a new avenue for attaining prolonged tissue rentention for covalently attached drugs.

Coating nanoparticles with poly(ethylene glycol) (PEG) is widely used to achieve long‐circulating properties after infusion. While PEG reduces binding of opsonins to the particle surface, immunogenic anti‐PEG side‐effects show that PEGylated nanoparticles are not truly “stealth” to surface active proteins. A major obstacle for understanding the complex interplay between opsonins and nanoparticles is the averaging effects of the bulk assays that are typically applied to study protein adsorption to nanoparticles. Here, a microscopy‐based method for directly quantifying opsonization at the single nanoparticle level is presented. Various surface coatings are investigated on liposomes, including PEG, and show that opsonization by both antibodies and complement C3b is highly dependent on the surface chemistry. It is further demonstrated that this opsonization is heterogeneous, with opsonized and non‐opsonized liposomes co‐existing in the same ensemble. Surface coatings modify the percentage of opsonized liposomes and/or opsonin surface density on the liposomes, with strikingly different patterns for antibodies and complement. Thus, this assay provides mechanistic details about opsonization at the single nanoparticle level previously inaccessible to established bulk assays.

Background: The accumulation of liposome encapsulated chemotherapy in solid cancers is dependent on the presence of the enhanced permeability and retention (EPR) effect. Positron emission tomography (PET) imaging with a liposome encapsulated radioisotope, such as liposome encapsulated Cu-64 (⁶⁴Cu-liposome) may help to identify tumors with high liposome accumulation, and thereby stratify patients based on expected benefit from liposomal chemotherapy. However, intravenous administration of liposomes without a cytotoxic content is complicated by the accelerated blood clearance (ABC) phenomenon for succeeding therapeutic liposome dosing. Alternative markers for assessing the tumor’s EPR level are therefore warranted. Materials and Methods: To increase our understanding of EPR variations and to ultimately identify an alternative marker for the EPR effect, we investigated the correlation between ⁶⁴Cu-liposome PET/CT (EPR effect) and ⁶⁸Ga-RGD PET/CT (neoangiogenesis), ¹⁸F-FDG PET/CT (glycolysis), diffusion-weighted MRI (diffusivity) and interstitial fluid pressure in two experimental cancer models (CT26 and COLO 205). Results: ⁶⁴Cu-liposome and ⁶⁸Ga-RGD SUV_max displayed a significant moderate correlation, however, none of the other parameters evaluated displayed significant correlations. These results indicate that differences in neoangiogenesis may explain some EPR variability, however, as correlations were only moderate and not observed for SUV_mean, ⁶⁸Ga-RGD is probably insufficient to serve as a stand-alone surrogate marker for quantifying the EPR effect and stratifying patients.

Immunosuppressive cells in the tumor microenvironment allow cancer cells to escape immune recognition and support cancer progression and dissemination. To improve therapeutic efficacy, we designed a liposomal oxaliplatin formulation (PCL8-U75) that elicits cytotoxic effects toward both cancer and immunosuppressive cells via protease-mediated, intratumoral liposome activation. The PCL8-U75 liposomes displayed superior therapeutic efficacy across all syngeneic cancer models in comparison to free-drug and liposomal controls. The PCL8-U75 depleted myeloid-derived suppressor cells and tumor-associated macrophages in the tumor microenvironment. The combination of improved cancer cell cytotoxicity and depletion of immunosuppressive populations of immune cells is attractive for combination with immune-activating therapy. Combining the PCL8-U75 liposomes with a TLR7 agonist induced immunological rejection of established tumors. This combination therapy increased intratumoral numbers of cancer antigen–specific cytotoxic T cells and Foxp3⁻ T helper cells. These results are encouraging toward advancing liposomal drug delivery systems with anticancer and immune-modulating properties into clinical cancer therapy.

The liquid-based Carbo-gel technology enables real-time multimodal image guidance for improved therapeutic precision.