Bart J. Crielaard

Nanomedicines for Inflammatory Arthritis: Head-to-Head Comparison of Glucocorticoid-Containing Polymers, Micelles, and Liposomes

As an emerging research direction, nanomedicine has been increasingly utilized to treat inflammatory diseases. In this head-to-head comparison study, four established nanomedicine formulations of dexamethasone, including liposomes (L-Dex), core-cross-linked micelles (M-Dex), slow releasing polymeric prodrugs (P-Dex-slow), and fast releasing polymeric prodrugs (P-Dex-fast), were evaluated in an adjuvant-induced arthritis rat model with an equivalent dose treatment design. It was found that after a single i.v. injection, the formulations with the slower drug release kinetics (i.e., M-Dex and P-Dex-slow) maintained longer duration of therapeutic activity than those with relatively faster drug release kinetics, resulting in better joint protection. This finding will be instructional in the future development and optimization of nanomedicines for the clinical management of rheumatoid arthritis. The outcome of this study also illustrates the value of such head-to-head comparison studies in translational nanomedicine research.

Glucocorticoid‐Loaded Core‐Cross‐Linked Polymeric Micelles with Tailorable Release Kinetics for Targeted Therapy of Rheumatoid Arthritis

Polymerizable and hydrolytically cleavable dexamethasone (DEX, red dot in picture) derivatives were covalently entrapped in core-cross-linked polymeric micelles that were prepared from a thermosensitive block copolymer (yellow and gray building block). By varying the oxidation degree of the thioether in the drug linker, the release rate of DEX could be controlled. The DEX-loaded micelles were used for efficient treatment of inflammatory arthritis in two animal models.

Distinct roles for hepcidin and interleukin-6 in the recovery from anemia in mice injected with heat-killed Brucella abortus

Anemia of inflammation (AI) is commonly observed in chronic inflammatory states and may hinder patient recovery and survival. Induction of hepcidin, mediated by interleukin 6, leads to iron-restricted erythropoiesis and anemia. Several translational studies have been directed at neutralizing hepcidin overexpression as a therapeutic strategy against AI. However, additional hepcidin-independent mechanisms contribute to AI, which are likely mediated by a direct effect of inflammatory cytokines on erythropoiesis. In this study, we used wild-type, hepcidin knockout (Hamp-KO) and interleukin 6 knockout (IL-6-KO) mice as models of AI. AI was induced with heat-killed Brucella abortus (BA). The distinct roles of iron metabolism and inflammation triggered by interleukin 6 and hepcidin were investigated. BA-treated wild-type mice showed increased expression of hepcidin and inflammatory cytokines, as well as transitory suppression of erythropoiesis and shortened red blood cell lifespan, all of which contributed to the severe anemia of these mice. In contrast, BA-treated Hamp-KO or IL-6-KO mice showed milder anemia and faster recovery compared with normal mice. Moreover, they exhibited different patterns in the development and resolution of anemia, supporting the notion that interleukin 6 and hepcidin play distinct roles in modulating erythropoiesis in AI.

A biodegradable antibiotic delivery system based on poly-(trimethylene carbonate) for the treatment of osteomyelitis

Background and purpose Many investigations on biodegradable materials acting as an antibiotic carrier for local drug delivery are based on poly(lactide). However, the use of poly(lactide) implants in bone has been disputed because of poor bone regeneration at the site of implantation. Poly(trimethylene carbonate) (PTMC) is an enzymatically degradable polymer that does not produce acidic degradation products. We explored the suitability of PTMC as an antibiotic releasing polymer for the local treatment of osteomyelitis. Methods This study addressed 2 separate attributes of PTMC: (1) the release kinetics of gentamicin-loaded PTMC and (2) its behavior in inhibiting biofilm formation. Both of these characteristics were compared with those of commercially available gentamicin-loaded poly(methylmethacrylate) (PMMA) beads, which are commonly used in the local treatment of osteomyelitis. Results In a lipase solution that mimics the in vivo situation, PTMC discs with gentamicin incorporated were degraded by surface erosion and released 60% of the gentamicin within 14 days. This is similar to the gentamicin release from clinically used PMMA beads. Moreover, biofilm formation by Staphylococcus aureus was inhibited by approximately 80% over at least 14 days in the presence of gentamicin-loaded PTMC discs. This is similar to the effect of gentamicin-loaded PMMA beads. In the absence of the lipase, surface erosion of PTMC discs did not occur and gentamicin release and biofilm inhibition were limited. Interpretation Since gentamicin-loaded PTMC discs show antibiotic release characteristics and biofilm inhibition characteristics similar to those of gentamicin-loaded PMMA beads, PTMC appears to be a promising biodegradable carrier in the local treatment of osteomyelitis.

Screening of budesonide nanoformulations for treatment of inflammatory bowel disease in an inflamed 3D cell-culture model

Drug formulation screenings for treatment of inflammatory bowel disease (IBD) are mostly conducted in chemically induced rodent models that represent acute injury-caused inflammation instead of a chronic condition. To accurately screen drug formulations for chronic IBD, a relevant model that mimics the chronic condition in vitro is urgently needed. In an effort to reduce and potentially replace this scientifically and ethically questionable animal testing for IBD drugs, our laboratory has developed an in vitro model for the inflamed intestinal mucosa observed in chronic IBD, which allows high-throughput screening of anti-inflammatory drugs and their formulations. The in vitro model consists of intestinal epithelial cells, human blood-derived macrophages, and dendritic cells that are stimulated by the inflammatory cytokine interleukin-1β. In this study, the model was utilized for evaluation of the efficacy and deposition of budesonide, an anti-inflammatory drug, in three different pharmaceutical formulations: (1) a free drug solution, (2) encapsulated into PLGA nanoparticles, and (3) encapsulated into liposomes. The in vitro model of the inflamed intestinal mucosa demonstrated its ability to differentiate therapeutic efficacy among the formulations while maintaining the convenience of conventional in vitro studies and adequately representing the complex pathophysiological changes observed in vivo.

Liposomal corticosteroids for the treatment of inflammatory disorders and cancer

Glucocorticoids (GC) are known for their potent immunosuppressive and anti-inflammatory properties. As a consequence, they have been extensively used for the treatment of many different diseases. Prolonged and/or high-dose GC therapy, however, generally comes with severe side effects, resulting not only from their very diverse mechanism(s) of action, but also from their relatively poor biodistribution. Drug delivery systems, and in particular liposomes, have been extensively used to enhance the biodistribution and the target site accumulation of GC, and to thereby improve the balance between their efficacy and their toxicity. Many different types of liposomes have been employed, and both local and systemic treatments have been evaluated. We here summarize the progress made in the use of liposomal GC formulations for the treatment of asthma, rheumatoid arthritis, multiple sclerosis and cancer, and we show that the targeted delivery of GC to pathological sites holds significant clinical potential.

Macrophages and liposomes in inflammatory disease : Friends or foes?

Liposome-encapsulated corticosteroids have shown to exert strong beneficial effects in inflammatory diseases, such as arthritis and cancer. To extend the clinical applicability of these potent nanomedicines, the therapeutic effect of dexamethasone phosphate loaded long-circulating liposomes (LCL-DXP) was evaluated in animal models of multiple sclerosis (MS) and Crohns disease (CD). In mice with experimental autoimmune encephalitis (EAE), a model for MS, treatment with LCL-DXP, but not free DXP, resulted in a decrease in disease activity when compared to PBS treated mice. In contrast, in mice with chronic DSS-induced colitis, a model for CD, treatment with LCL-DXP did not induce an improvement, but in fact worsened the fecal blood loss after treatment, indicating an aggravation of the disease. It is hypothesized that modulation of macrophage polarization towards a M2 phenotype underlies the efficacy of corticosteroid-based drug delivery systems, which is supported by the presented data. On the one hand, M1 polarized macrophages are part of the pathogenesis of MS; the modulation to M2-polarization by LCL-DXP is therefore beneficial. On the other hand, M1-polarized intestinal macrophages fulfill a protective and inflammation-suppressing role in intestinal homeostasis; changing their phenotype to M2 causes reduced protection to invading microorganisms, leading to a more severe intestinal inflammation. These findings therefore indicate that the interplay between the specific phenotype of macrophages and the specific inflammatory context of the inflammatory disease in question may be an important determining factor in the therapeutic applicability of liposomal corticosteroids in inflammatory disease.

MRI evaluation of the antitumor activity of paramagnetic liposomes loaded with prednisolone phosphate

The design of long circulating liposomes co-loaded with the glucocorticoid prednisolone phosphate (PLP) and the amphiphilic paramagnetic contrast agent Gd-DOTAMA(C(18))(2) allowed the MRI-guided in vivo visualization of the delivery and biodistribution of PLP, as well as the monitoring of drug efficacy. The performance of this theranostic probe was investigated in a mouse model bearing a melanoma B16 syngeneic tumor. The release kinetics of the drug were evaluated in vitro where it displayed a peculiar behavior characterized by a fast process (completed in few hours) involving only a small portion (<5%) of the drug. Interestingly, the incorporation of the amphiphilic imaging reporter in the liposomal bilayer slightly increased the amount of the fast-release portion (<10%), thus suggesting that it could be attributed to a drug fraction embedded in the liposomal bilayer. In fact, the release of a hydrophilic imaging probe encapsulated in the inner core of the same long circulating liposomes formulated for carrying the drug, displayed different, single-step, kinetics. The in vivo monitoring of the antitumor activity of the nanomedicine revealed that the incorporation of the MRI probe into the liposome bilayer did not significantly affect the drug efficacy. The in vivo experiments also indicated a relevant and fast liposome uptake from macrophage-rich organs like spleen and liver, which reduced the tumor accumulation of the liposomes. The accumulation of the amphipatic MRI label caused the occurrence of a long-term residual T(1) contrast still detectable 1week after injection.

Targeting iron metabolism in drug discovery and delivery

Iron fulfils a central role in many essential biochemical processes in human physiology; thus, proper processing of iron is crucial. Although iron metabolism is subject to relatively strict physiological control, numerous disorders, such as cancer and neurodegenerative diseases, have recently been linked to deregulated iron homeostasis. Consequently, iron metabolism constitutes a promising and largely unexploited therapeutic target for the development of new pharmacological treatments for these diseases. Several iron metabolism-targeted therapies are already under clinical evaluation for haematological disorders, and these and newly developed therapeutic agents are likely to have substantial benefit in the clinical management of iron metabolism-associated diseases, for which few efficacious treatments are currently available.

Targeted delivery of small interfering RNA to angiogenic endothelial cells with liposome-polycation-DNA particles

Angiogenesis is an attractive target for cancer therapy, due to its central position in tumor growth and development. Vascular Endothelial Growth Factor (VEGF) and its receptors (VEGFRs) play a key role in the angiogenic process. A promising strategy for targeting VEGF-mediated angiogenesis is RNA interference (RNAi) using short interfering RNA (siRNA). However, for efficacious RNAi a well-designed siRNA delivery system is crucial. Liposome-Polycation-DNA (LPD) particles form a promising system for siRNA delivery to tumors. In order to target angiogenic endothelial cells, LPD particles may be modified with a targeting ligand, such as a cyclic Arg-Gly-Asp (RGD) peptide that specifically binds to integrins expressed on tumor-associated endothelial cells. In the current study, RGD-targeted PEGylated LPD particles containing VEGFR-2 siRNA were prepared and optimized with respect to their size and charge by varying protamine content, carrier DNA content for stronger complexation, and PEGylation density. The size of the optimized particles was around 200 nm and the ζ-potential was approximately +20 mV. The uptake and silencing efficacy of the RGD-targeted PEGylated LPD particles were evaluated in H5V cells (murine endothelial cells) and Human Umbilical Vein Endothelial cells (HUVECs). When compared to non-targeted LPD particles, enhanced uptake and silencing of VEGFR-2 expression was observed for RGD-targeted PEGylated LPD particles. In conclusion, the RGD-targeted PEGylated LPD particles containing VEGFR-2 siRNA presented here may be a promising approach for targeting VEGF-mediated angiogenesis in cancer therapy.