Phospholipid bilayers that constitute endo-lysosomal vesicles can pose a barrier to delivery of biologic drugs to intracellular targets. To overcome this barrier, a number of synthetic drug carriers ...have been engineered to actively disrupt the endosomal membrane and deliver cargo into the cytoplasm. Here, we describe the hemolysis assay, which can be used as rapid, high-throughput screen for the cytocompatibility and endosomolytic activity of intracellular drug delivery systems. In the hemolysis assay, human red blood cells and test materials are co-incubated in buffers at defined pHs that mimic extracellular, early endosomal, and late endo-lysosomal environments. Following a centrifugation step to pellet intact red blood cells, the amount of hemoglobin released into the medium is spectrophotometrically measured (405 nm for best dynamic range). The percent red blood cell disruption is then quantified relative to positive control samples lysed with a detergent. In this model system the erythrocyte membrane serves as a surrogate for the lipid bilayer membrane that enclose endo-lysosomal vesicles. The desired result is negligible hemolysis at physiologic pH (7.4) and robust hemolysis in the endo-lysosomal pH range from approximately pH 5-6.8.
Coupled systems of in vitro microfabricated organs-on-a-chip containing small populations of human cells are being developed to address the formidable pharmacological and physiological gaps between ...monolayer cell cultures, animal models, and humans that severely limit the speed and efficiency of drug development. These gaps present challenges not only in tissue and microfluidic engineering, but also in systems biology: how does one model, test, and learn about the communication and control of biological systems with individual organs-on-chips that are one-thousandth or one-millionth of the size of adult organs, or even smaller, i.e., organs for a milliHuman (mHu) or microHuman (μHu)? Allometric scaling that describes inter-species variation of organ size and properties provides some guidance, but given the desire to utilize these systems to extend and validate human pharmacokinetic and pharmacodynamic (PK/PD) models in support of drug discovery and development, it is more appropriate to scale each organ functionally to ensure that it makes the suitable physiological contribution to the coupled system. The desire to recapitulate the complex organ-organ interactions that result from factors in the blood and lymph places a severe constraint on the total circulating fluid (~5 mL for a mHu and ~5 μL for a μHu) and hence on the pumps, valves, and analytical instruments required to maintain and study these systems. Scaling arguments also provide guidance on the design of a universal cell-culture medium, typically without red blood cells. This review presents several examples of scaling arguments and discusses steps that should ensure the success of this endeavour.
Peptides and biologics provide unique opportunities to modulate intracellular targets not druggable by conventional small molecules. Most peptides and biologics are fused with cationic uptake ...moieties or formulated into nanoparticles to facilitate delivery, but these systems typically lack potency due to low uptake and/or entrapment and degradation in endolysosomal compartments. Because most delivery reagents comprise cationic lipids or polymers, there is a lack of reagents specifically optimized to deliver cationic cargo. Herein, we demonstrate the utility of the cytocompatible polymer poly(propylacrylic acid) (PPAA) to potentiate intracellular delivery of cationic biomacromolecules and nano-formulations. This approach demonstrates superior efficacy over all marketed peptide delivery reagents and enhances delivery of nucleic acids and gene editing ribonucleoproteins (RNPs) formulated with both commercially-available and our own custom-synthesized cationic polymer delivery reagents. These results demonstrate the broad potential of PPAA to serve as a platform reagent for the intracellular delivery of cationic cargo.
Endolysosome entrapment is one of the key barriers to the therapeutic use of biologic drugs that act intracellularly. The screening of prospective nanoscale endosome-disrupting delivery technologies ...is currently limited by methods that are indirect and cumbersome. Here, we statistically validate Galectin 8 (Gal8) intracellular tracking as a superior approach that is direct, quantitative, and predictive of therapeutic cargo intracellular bioactivity through in vitro high-throughput screening and in vivo validation. Gal8 is a cytosolically dispersed protein that, when endosomes are disrupted, redistributes by binding to glycosylation moieties selectively located on the inner face of endosomal membranes. The quantitative redistribution of a Gal8 fluorescent fusion protein from the cytosol into endosomes is demonstrated as a real-time, live-cell assessment of endosomal integrity that does not require labeling or modification of either the carrier or the biologic drug and that allows quantitative distinction between closely related, endosome-disruptive drug carriers. Through screening two families of siRNA polymeric carrier compositions at varying dosages, we show that Gal8 endosomal recruitment correlates strongly (r = 0.95 and p < 10–4) with intracellular siRNA bioactivity. Through this screen, we gathered insights into how composition and molecular weight affect endosome disruption activity of poly(ethylene glycol)-b-(2-(dimethylamino)ethyl methacrylate)-co-(butyl methacrylate) PEG-(DMAEMA-co-BMA) siRNA delivery systems. Additional studies showed that Gal8 recruitment predicts intracellular bioactivity better than current standard methods such as Lysotracker colocalization (r = 0.35, not significant), pH-dependent hemolysis (not significant), or cellular uptake (r = 0.73 and p < 10–3). Importantly, the Gal8 recruitment method is also amenable to fully objective high-throughput screening using automated image acquisition and quantitative image analysis, with a robust estimated Z′ of 0.6 (whereas assays with Z′ > 0 have high-throughput screening utility). Finally, we also provide measurements of in vivo endosomal disruption based on Gal8 visualization (p < 0.03) of a nanocarrier formulation confirmed to produce significant cytosolic delivery and bioactivity of siRNA within tumors (p < 0.02). In sum, this report establishes the utility of Gal8 subcellular tracking for the rapid optimization and high-throughput screening of the endosome disruption potency of intracellular delivery technologies.
OBJECTIVE The objective of this study was to detect 5-aminolevulinic acid (ALA)-induced tumor fluorescence from glioma below the surface of the surgical field by using red-light illumination. METHODS ...To overcome the shallow tissue penetration of blue light, which maximally excites the ALA-induced fluorophore protoporphyrin IX (PpIX) but is also strongly absorbed by hemoglobin and oxyhemoglobin, a system was developed to illuminate the surgical field with red light (620-640 nm) matching a secondary, smaller absorption peak of PpIX and detecting the fluorescence emission through a 650-nm longpass filter. This wide-field spectroscopic imaging system was used in conjunction with conventional blue-light fluorescence for comparison in 29 patients undergoing craniotomy for resection of high-grade glioma, low-grade glioma, meningioma, or metastasis. RESULTS Although, as expected, red-light excitation is less sensitive to PpIX in exposed tumor, it did reveal tumor at a depth up to 5 mm below the resection bed in 22 of 24 patients who also exhibited PpIX fluorescence under blue-light excitation during the course of surgery. CONCLUSIONS Red-light excitation of tumor-associated PpIX fluorescence below the surface of the surgical field can be achieved intraoperatively and enables detection of subsurface tumor that is not visualized under conventional blue-light excitation. Clinical trial registration no.: NCT02191488 (clinicaltrials.gov).
Abstract This study utilized a national database to evaluate 90 day postoperative complication rates after total knee arthroplasty (TKA) in super obese (BMI > 50 kg/m2 ) patients ( n = 7666) compared ...to non-obese patients ( n = 1,212,793), obese patients ( n = 291,914), morbidly obese patients ( n = 169,308) and revision TKA patients ( n = 28,812). Super obese patients had significantly higher rates of local and systemic complications compared to all other BMI groups as well as those undergoing revision TKA with higher rates of venous thromboembolism (VTE), infection, and medical complications. Super obesity is associated with dramatically increased rates of postoperative complications after TKA compared to non-obese, obese, and morbidly obese patients as well as those undergoing revision TKA.
Purpose This study aimed to (1) evaluate the ex vivo restoration of the long head biceps length-tension for both arthroscopic suprapectoral biceps tenodesis (ASPBT) and open subpectoral biceps ...tenodesis (OSPBT) techniques and (2) assess how location in the proximal humerus affects pullout strength for tenodesis using an interference screw implant. Methods Eighteen matched cadaveric shoulders were randomized to OSPBT or ASPBT groups (9 each). Tenodesis was performed using clinical techniques. Preoperatively, a metallic bead was placed in the biceps tendon and a fluoroscopic image was obtained. Postoperatively, an image was obtained to evaluate the location of the tenodesis and the metallic bead and determine tensioning. Biomechanical load-to-failure testing was then performed. Results The ASPBT technique resulted in an average of 2.15 ± 0.62 cm of biceps overtensioning compared with 0.78 ± 0.35 cm ( P < .001) in the OSPBT group. The average load to failure in the ASPBT group was 138.8 ± 29.1 N compared with 197 ± 38.6 N ( P = .002) in the OSPBT group. Failure caused by implant pullout was significantly more frequent in the ASPBT group (7 of 9) than in the OSPBT group (1 of 9). Conclusions The described ASPBT technique using an interference screw implant has the tendency to overtension the biceps and has a significantly decreased ultimate load to failure compared with an open subpectoral technique in matched cadaveric specimens. Clinical Relevance This study shows differences in the biomechanical properties of OSPBT and ASPBT. Modification of currently published ASPBT techniques may be necessary to improve restoration of the physiological length-tension relationship of the biceps. Clinical studies may need to clarify if the lower ultimate load to failure for the ASPBT technique is clinically significant.
Background:
While a vast body of literature exists describing biceps tenodesis techniques and evaluating the biomechanical aspects of tenodesis locations or various implants, little literature ...presents useful clinical outcomes to guide surgeons in their decision to perform a particular method of tenodesis.
Purpose/Hypothesis:
To compare the clinical outcomes of open subpectoral biceps tenodesis (OSPBT) and arthroscopic suprapectoral biceps tenodesis (ASPBT). Our null hypothesis was that both methods would yield satisfactory results with regard to shoulder and biceps function, postoperative shoulder scores, pain relief, and complications.
Study Design:
Cohort study; Level of evidence, 3.
Methods:
Patients who underwent either ASPBT or OSPBT for isolated superior labrum or long head of the biceps lesions with a minimum follow-up of 2 years were evaluated with several validated clinical outcome measures and physical examinations including range of motion and strength.
Results:
Between 2007 and 2011, a total of 82 patients met all inclusion and exclusion criteria, which included 32 patients with ASPBT and 50 patients with OSPBT; 27 of 32 (84.4%) patients with ASPBT and 35 of 50 (70.0%) patients with OSPBT completed clinical follow-up. Overall outcomes for both procedures were satisfactory. No significant differences were noted in postoperative Constant-Murley (ASPBT: 90.7; OSPBT: 91.8; P = .755), American Shoulder and Elbow Surgeons (ASPBT: 90.1; OSPBT: 88.4; P = .735), Single Assessment Numeric Evaluation (ASPBT: 87.4; OSPBT: 86.8; P = .901), Simple Shoulder Test (ASPBT: 10.4; OSPBT: 10.6; P = .762), long head of the biceps (ASPBT: 91.6; OSPBT: 93.6; P = .481), or Veterans RAND 36-Item Health Survey (ASPBT: 81.0; OSPBT: 80.1; P = .789) scores. No significant range of motion or strength differences was noted between the procedures.
Conclusion:
Both ASPBT and OSPBT yield excellent clinical and functional results for the management of isolated superior labrum or long head of the biceps lesions. No significant differences in clinical outcomes as determined by several validated outcome measures were found between the 2 tenodesis methods, nor were any significant range of motion or strength deficits noted at a minimum 2 years postoperatively.
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