Interfacial Pressure and Viscoelasticity of Antibodies and Their Correlation to Long-Term Stability in Formulation.

Monoclonal antibodies (mAbs) form viscoelastic gel-like layers at the air-water interface due to their amphiphilic nature, and this same protein characteristic can lead to undesired aggregation of proteins in therapeutic formulations. We hypothesize that the interfacial viscoelasticity and surface pressure of mAbs at the air-water interface will correlate with their long-term stability. To test this hypothesis, the interfacial viscoelastic rheology and surface pressure of five different antibodies with varying visible particle counts from a three-year stability study were measured. We find that both the surface pressures and interfacial elastic moduli correlate well with the long-time mAb solution stability within a class of mAbs with the interfacial elastic moduli being particularly sensitive to discriminate between stable and unstable mAbs across a range of formulations. Furthermore, X-ray reflectivity was used to gain insight into the interfacial structure of mAbs at the air-water interface, providing a possible molecular mechanism to explain the relationship between interfacial elastic moduli and the long-term stability.

The Promising Therapeutic Potential of Oligonucleotides for Pulmonary Fibrotic Diseases.

INTRODUCTION: Fibrotic lung diseases represent a large subset of diseases with an unmet clinical need. Oligonucleotide therapies (ONT) are a promising therapeutic approach for the treatment of pulmonary disease as they can inhibit pathways that are otherwise difficult to target. Additionally, targeting the lung specifically with ONT is advantageous because it reduces the possibilities of systemic side effects and tolerability concerns. AREAS COVERED: This review presents the chemical basis of designing various ONTs currently known to treat fibrotic lung diseases. Further, the authors have also discussed the delivery vehicle, routes of administration, physiological barriers of the lung, and toxicity concerns with ONTs. EXPERT OPINION: ONTs provide a promising therapeutic approach for the treatment of fibrotic diseases of the lung, particularly because ONTs directly delivered to the lung show little systemic side effects compared to current therapeutic strategies. Dry powder aerosolized inhalers may be a good strategy for getting ONTs into the lung in humans. However, as of now, no dry powder ONTs have been approved for use in the clinical setting, and this challenge must be overcome for future therapies. Various delivery methods that can aid in direct targeting may also improve the use of ONTs for lung fibrotic diseases.

Structure-function relationships of cholesterol mobilization from the endo-lysosome compartment of NPC1-deficient human cells by ß-CD polyrotaxanes.

Niemann-Pick Type C is a rare metabolic disorder characterized by the cellular accumulation of cholesterol within endosomal and lysosomal compartments. 2-Hydroxypropyl-ß-cyclodextrin (HP-ß-CD) containing polyrotaxanes represent an attractive approach for treating this disease due to their ability to circulate in the blood stream for longer periods of time as a prodrug form of HP-ß-CD. Once inside the cell, the macromolecular structure is thought to break down into the Pluronic precursor and the active cyclodextrin agent that promotes cholesterol mobilization from the aberrant accumulations within NPC-deficient cells. We now report that both cholesterol and decaarginine (R10) endcapped polyrotaxanes are able to remove cholesterol from NPC1 patient fibroblasts. R10 endcapped materials enter these cells and are localized within endosomes after 16 h. The cholesterol mobilization from endo-lysosomal compartments of NPC1 cells by the polyrotaxanes was directly related to their extent of endcapping and their threading efficiency. Incorporation of 4-sulfobutylether-ß-cyclodextrin (SBE-ß-CD) significantly improved cholesterol mobilization due to the improved solubility of the compounds. Additionally, in our efforts to scale-up the synthesis for preclinical studies, we prepared a library of polyrotaxanes using a solid phase synthesis method. These compounds also led to significant cholesterol mobilization from the cells, however, cytotoxicity studies showed that they were substantially more toxic than those prepared by the solvent-assisted method, thus limiting the therapeutic utility of agents prepared by this expedited method. Our findings demonstrate that complete endcapping of the polyrotaxanes and improved solubility are important design features for delivering high copy numbers of therapeutic ß-CD to promote enhanced sterol clearance in human NPC1-deficient cells.

Immunostimulatory Response of RWFV Peptide-Targeted Lipid Nanoparticles on Bladder Tumor Associated Cells.

Bladder carcinoma is the most expensive tumor type to treat on a cost-per-patient basis from diagnosis to death. Treatment with Bacillus Calmette Guerin (BCG) instillation is the only approved immunotherapy in the clinic for the remission of superficial bladder carcinoma. Unfortunately, frequent relapses, high local morbidity, risk of systemic mycobacterial infection, and occasional supply chain interruptions limit the utility of BCG for bladder cancer treatment. It is well known that BCG utilizes an adhesin protein known as fibronectin attachment protein that possesses a crucial RWFV peptide sequence for binding to the bladder tumor microenvironment prior to the initiation of the immunotherapeutic response. We report a RWFV-targeted, pH-responsive stabilized lipid nucleic acid nanoparticle (LNP) vehicle for the effective delivery of an immunotherapeutic oligonucleotide, CpG, that is assembled using a glass microfluidic Chemtrix 3221 reactor. Our small-angle X-ray scattering studies revealed a layer-by-layer assembly of the oligonucleotides with a repeat distance of 6.04 nm within the LNP. Using flow cytometry to evaluate the different cell types found in the bladder tumor microenvironment, RWFV-targeted LNPs were found to attach specifically to fibronectin-secreting cells in culture during a 2 h incubation period. The trafficking and cellular fate of these targeted LNPs were revealed by confocal microscopy of RAW264.7 macrophages to enter the endocytotic pathway within 4 h post treatment. Importantly, control studies reveal that only the pH-sensitive LNP formulation is capable of efficiently releasing the payload within 12 h. As a result, the targeted pH-sensitive LNP resulted in higher expression levels of costimulatory molecules CD83, CD 86, and MHC II, while also inducing higher levels of TNF-α secretion from macrophages. These results demonstrate that RWFV-targeted, pH-sensitive LNP formulations are capable of maximum immunotherapeutic response, potentially making them a highly efficient, lower risk, and readily manufactured alternative to BCG immunotherapy.

Development And In Vitro Characterization Of Bladder Tumor Cell Targeted Lipid-Coated Polyplex For Dual Delivery Of Plasmids And Small Molecules.

BACKGROUND: Bladder cancer is the fourth most common cancer in men and eleventh most common in women. Combination therapy using a gene and chemotherapeutic drug is a potentially useful strategy for treating bladder cancer in cases where a synergistic benefit can be achieved successfully. This approach relies on developing drug combinations using carrier systems that can load both hydrophilic genes and hydrophobic drugs. Ideally, the formulation for carrier system should be free of traditional high shear techniques such as sonication and extrusion to reduce shear-induced nucleic acid strand breakage. Moreover, the system should be able to protect the nucleic acid from enzymatic attack and deliver it specifically to the tumor site. MATERIALS AND METHODS: A dual payload carrier system that was formulated using a simple flow mixing technique to complex anionic plasmid (EGFP-NLS) using a cationic polymer (CD-PEI2.5kD) followed by coating of the polyplex using lipid membranes. The resulting lipid-coated polyplex (LCP) formulations are targeted to bladder cancer cells by employing a bacterial adhesive peptide sequence, RWFV, that targets the LCP to the tumor stroma for efficiently delivering reporter plasmid, EGFP-NLS and a model small molecule drug, pyrene, to the cancer cells. RESULTS: Encapsulation efficiency of the peptide targeted carrier for the plasmid was 50% ± 0.4% and for pyrene it was 16% ± 0.4%. The ability of the targeted LCP to transfect murine bladder cancer cells was 4-fold higher than LCP bearing a scrambled peptide sequence. Fluorescence of cells due to pyrene delivery was highest after 4 hrs using targeted LCP. Finally, we loaded the peptide targeted LCP with anti-cancer agent, curcumin. The targeted formulation of curcumin resulted in only 45% viable cancer cells at a concentration of 5 µg/mL, whereas the empty and non-targeted formulations did not result any significant cell death. CONCLUSION: These results demonstrate the specificity of the targeting peptide sequence in engaging tumor cells and the utility of the developed carrier platform to deliver a dual payload to bladder tumor cells.

Synthesis of the Anionic Hydroxypropyl-ß-cyclodextrin:Poly(decamethylenephosphate) Polyrotaxane and Evaluation of its Cholesterol Efflux Potential in Niemann-Pick C1 Cells.

Niemann-Pick type C disease (NPC) is a lysosomal storage disease that is characterized by a progressive accumulation of unesterified cholesterol in the lysosomes leading to organ damage from cell dysfunction. Hydroxypropyl-ß-cyclodextrin (HP-ß-CD) is an attractive drug candidate for treating NPC, as it diminishes cholesterol accumulation in NPC cells. Systemic HP-ß-CD treatment, however, is limited by rapid renal clearance. We designed a new anionic HP-ß-CD polyrotaxane to act as a slow release formulation based on a polyalkylene phosphate core to improve the pharmacokinetics. The polyalkylene phosphate comprises hydrophobic decamethylene spacers linked by biodegradable anionic phosphodiester bonds. HP-ß-CD was threaded onto this polymer first and α-CD afterwards to prevent burst release of the threaded HP-ß-CD. Our findings show that HP-ß-CD was slowly released from the watersoluble polyrotaxane over a 30 days period. The polyrotaxane provided persistently diminished cholesterol levels in NPC1 cells by 20% relative to untreated cells. These results demonstrate the therapeutic potential of this novel HP-ß-CD polyrotaxane for the mobilization of aberrantly stored cholesterol in NPC1 cells.

An Implantable Ultrasonically-Powered Micro-Light-Source (µLight) for Photodynamic Therapy.

Photodynamic therapy (PDT) is a promising cancer treatment modality that can selectively target unresectable tumors through optical activation of cytotoxic agents, thus reducing many side effects associated with systemic administration of chemotherapeutic drugs. However, limited light penetration into most biological tissues have so far prevented its widespread adoption beyond dermatology and a few other oncological applications in which a fiber optic can be threaded to the desired locations via an endoscopic approach (e.g., bladder). In this paper, we introduce an ultrasonically powered implantable microlight source, µLight, which enables in-situ localized light delivery to deep-seated solid tumors. Ultrasonic powering allows for small receiver form factor (mm-scale) and power transfer deep into the tissue (several centimeters). The implants consist of piezoelectric transducers measuring 2 × 2 × 2 mm3 and 2 × 4 × 2 mm3 with surface-mounted miniature red and blue LEDs. When energized with 185 mW/cm2 of transmitted acoustic power at 720 kHz, µLight can generate 0.048 to 6.5 mW/cm2 of optical power (depending on size of the piezoelectric element and light wavelength spectrum). This allows powering multiple receivers to a distance of 10 cm at therapeutic light output levels (a delivery of 20-40 J/cm2 light radiation dose in 1-2 hours). In vitro tests show that HeLa cells irradiated with µLights undergo a 70% decrease in average cell viability as compared to the control group. In vivo tests in mice implanted with 4T1-induced tumors (breast cancer) show light delivery capability at therapeutic dose levels. Overall, results indicate implanting multiple µLights and operating them for 1-2 hours can achieve cytotoxicity levels comparable to the clinically reported cases using external light sources.

Fluorescent sensors for the basic metabolic panel enable measurement with a smart phone device over the physiological range.

The advanced functionality of portable devices such as smart phones provides the necessary hardware to potentially perform complex diagnostic measurements in any setting. Recent research and development have utilized cameras and data acquisition properties of smart phones to create diagnostic approaches for a variety of diseases or pollutants. However, in concentration measurements, such as blood glucose, the performance of handheld diagnostic devices depends largely on the sensing mechanism. To expand measurements to multiple components, often necessary in medical tests, with a single diagnostic device, robust platform based sensors are needed. Here, we developed a suite of dual wavelength fluorescent sensors with response characteristics necessary to measure each component of a basic metabolic panel, a common clinical measurement. Furthermore, the response of these sensors could be measured with a simple optical setup to convert a smart phone into a fluorescence measurement instrument. This approach could be used as a mobile basic metabolic panel measurement system for point of care diagnostics.