Impact of covalently Nile Red and covalently Rhodamine labeled fluorescent polymer micelles for the improved imaging of the respective drug delivery system.

Novel fluorescently labeled poly(ethylene glycol)-poly(hydroxyoctanoic acid) (MPEG-PHOA) block-copolymers were synthesized for the improved visualization of the deriving polymeric micelle drug delivery system. Albeit commonly used, one has to be aware that by simple incorporation of Nile Red (hydrophobic) or Rhodamine B (hydrophilic) as fluorescent compounds in nanocarriers (e.g., nanoparticles, liposomes or micelles) for imaging applications, these fluorescent probes can diffuse out of the carrier system and lead to artefacts due to the concomitant fluorescence loss or areal distribution. In order to inhibit such an uncontrolled diffusion, the Nile Red derivative 2-((9-(diethylamino)-5-oxo-5H-benzo[a]phenoxazin-2-yl)oxy)acetic acid was synthesized and covalently attached to the MPEG-PHOA block-copolymer via a mild Mitsunobu reaction to yield the desired MPEG-PHOA-Nile Red polymer for micelle preparations. Rhodamine B was coupled via its native carboxylic acid group with the copolymer MPEG-PHOA under mild conditions using DMAP, EDC, and NHS. For the proof of concept, aqueous solutions of composite micelles made of 0.5% (w/w fluorescence dye) MPEG-PHOA-dye and MPEG-PHOA copolymers were prepared ("spiking" of the non-labeled base MPEG-PHOA micelles) and characterized by transmission electron microscopy (TEM), dialysis and fluorescence spectrometry. The fluorescence intensity of the Nile Red in the solutions was followed up at physiological temperatures and pH values (37 °C, pH = 7.4 PBS buffer 0.01 M) over a period of 8 weeks. The labeled and non-labeled micelle formulations were tested in vitro in cells (Rhodamine-micelle formulations), then in vivo in a case study of an ophthalmic application (Nile Red micelle formulations). Both in vitro and in vivo experiments revealed a significant improvement of fluorescence stability of the MPEG-PHOA-dye formulations, facilitating the investigations on tracing the micelles and their stability. The results clearly demonstrate the value of the novel Nile Red and Rhodamine derivatives, whose simple synthesis and covalent attachment may easily be transferred to other nanosized polymeric drug delivery systems, e.g., MPEGylated or non-MPEGylated PLA/PLGA nanoparticles and be envisioned for novel theranostic systems.

Polymer-based nanoparticles loaded with a TLR7 ligand to target the lymph node for immunostimulation.

Small-molecule agonists for the Toll-like receptors (TLR) 7 and 8 are effective for the immunotherapy of skin cancer when used as topical agents. Their systemic use has however been largely unsuccessful due to dose-limiting toxicity. We propose a polymer-based nanodelivery system to target resiquimod, a TLR7 ligand, to the lymph node in order to focus the immunostimulatory activity and to prevent a generalized inflammatory response. We demonstrate successful encapsulation of resiquimod in methoxypoly(ethylene glycol)-b-poly(DL-lactic acid) (mPEG-PLA) and mixed poly(DL-lactic-co-glycolic acid) (PLGA)/mPEG-PLA nanoparticles. We show that these particles are taken up mainly by dendritic cells and macrophages, which are the prime initiators of anticancer immune responses. Nanoparticles loaded with resiquimod activate these cells, demonstrating the availability of the immune-stimulating cargo. The unloaded particles are non-inflammatory and do not have cytotoxic activity on immune cells. Following subcutaneous injection in mice, mPEG-PLA and PLGA/mPEG-PLA nanoparticles are detected in dendritic cells and macrophages in the draining lymph nodes, demonstrating the targeting potential of these particles. Thus, polymer-based nanoparticles represent a promising delivery system that allows lymph node targeting for small-molecule TLR7 agonists in the context of systemic cancer immunotherapy.

Synthesis of hydrophilic intra-articular microspheres conjugated to ibuprofen and evaluation of anti-inflammatory activity on articular explants.

The main limitation of current microspheres for intra-articular delivery of non-steroidal anti-inflammatory drugs (NSAIDs) is a significant initial burst release, which prevents a long-term drug delivery. In order to get a sustained delivery of NSAIDs without burst, hydrogel degradable microspheres were prepared by co-polymerization of a methacrylic derivative of ibuprofen with oligo(ethylene-glycol) methacrylate and poly(PLGA-PEG) dimethacrylate as degradable crosslinker. Microspheres (40-100 µm) gave a low yield of ibuprofen release in saline buffer (≈2% after 3 months). Mass spectrometry analysis confirmed that intact ibuprofen was regenerated indicating that ester hydrolysis occurred at the carboxylic acid position of ibuprofen. Dialysis of release medium followed by alkaline hydrolysis show that in saline buffer ester hydrolysis occurred at other positions in the polymer matrix leading to the release of water-soluble polymers (>6-8000 Da) conjugated with ibuprofen showing that degradation and drug release are simultaneous. By considering the free and conjugated ibuprofen, 13% of the drug is released in 3 months. In vitro, ibuprofen-loaded MS inhibited the synthesis of prostaglandin E2 in articular cartilage and capsule explants challenged with lipopolysaccharides. Covalent attachment of ibuprofen to PEG-hydrogel MS suppresses the burst release and allows a slow drug delivery for months and the cyclooxygenase-inhibition property of regenerated ibuprofen is preserved.

Intra-articular fate of degradable poly(ethyleneglycol)-hydrogel microspheres as carriers for sustained drug delivery.

A novel degradable microsphere (MS) for intra-articular drug delivery, composed of a polyethylene glycol (PEG) core containing degradable regions made of short poly-(lactic-co-glycolic acid) (PLGA) sequences - named PEG-hydrogel MS - was injected into the cavity of sheep shoulder joint, and compared to non-degradable MS devoid of hydrolysable crosslinker in terms of location, degradation and inflammation. One week after intra-articular injection both groups of MS were localized beneath the synovial lining of the synovial fringes located at bottom of the shoulder joint, while a fraction of particles remained in synovial fluid. Histological analyses made one and 4 weeks after intra-articular injection showed cell proliferation around the non-degradable MS entrapped within the synovium. By contrast, degradable PEG-hydrogel MS were surrounded by few cells. The degradation of degradable PEG-hydrogel MS within the synovium was slow and was not fully complete after four weeks. Our findings indicate that the tissue entrapment of MS below the synovial lining was independent of the material degradability, while degradable PEG-hydrogel MS are less inflammatory than the non-degradable one. Degradable PEG-hydrogel MS offer several advantages over the non-degradable MS as carriers for a sustained drug delivery in synovial tissue according to the low intensity of inflammatory reaction triggered in synovium.