In Vivo Imaging of Immune Rejection of MIN6 Cells Transplanted in C3H Mice.

Recently, we successfully utilized noninvasive magnetic resonance and bioluminescence imaging to track MIN6 cells subcutaneously transplanted in immunocompromised nude mice for up to 64 days. In this study, we further used bioluminescence imaging to investigate the immune rejection of MIN6 cells in immunocompetent C3H mice. A total of 5 × 106 luciferase-transfected MIN6 cells were implanted into the subcutaneous space of each nude or C3H mouse. After transplantation, hypoglycemia and persistent bioluminescence signals were observed in eight of eight (100%) nude mice and five of nine (56%) C3H mice (p < 0.05). We then presensitized a group of C3H mice with C57BL/6 spleen cells just prior to transplantation (n = 14). Interestingly, none of them had hypoglycemia or persistent bioluminescence signals (p < 0.01 vs. C3H mice without presensitization). Histological examination of the grafts revealed a lack or minimal presence of insulin-positive cells in recipients without hypoglycemia and persistent bioluminescence signals. In contrast, recipients with hypoglycemia and persistent bioluminescence signals showed a significant presence of insulin-positive cells in their grafts. Our results indicate that rejection of MIN6 cells occurred in C3H mice and could be enhanced by presensitization with C57BL/6 spleen cells and that bioluminescence imaging is a useful noninvasive tool for detecting rejection of subcutaneously transplanted MIN6 cells.

The Image-Histology Correlation of Subcutaneous mPEG-poly(Ala) Hydrogel-Embedded MIN6 Cell Grafts in Nude Mice.

Previously, we have successfully used noninvasive magnetic resonance (MR) and bioluminescence imaging to detect and monitor mPEG-poly(Ala) hydrogel-embedded MIN6 cells at the subcutaneous space for up to 64 days. In this study, we further explored the histological evolution of MIN6 cell grafts and correlated it with image findings. MIN6 cells were incubated overnight with chitosan-coated superparamagnetic iron oxide (CSPIO) and then 5 × 106 cells in the 100 µL hydrogel solution were injected subcutaneously into each nude mouse. Grafts were removed and examined the vascularization, cell growth and proliferation with anti-CD31, SMA, insulin and ki67 antibodies, respectively, at 8, 14, 21, 29 and 36 days after transplantation. All grafts were well-vascularized with prominent CD31 and SMA staining at all time points. Interestingly, insulin-positive cells and iron-positive cells were scattered in the graft at 8 and 14 days; while clusters of insulin-positive cells without iron-positive cells appeared in the grafts at 21 days and persisted thereafter, indicating neogrowth of MIN6 cells. Moreover, proliferating MIN6 cells with strong ki67 staining was observed in 21-, 29- and 36-day grafts. Our results indicate that the originally transplanted MIN6 cells proliferated from 21 days that presented distinctive bioluminescence and MR images.

Magnetic Resonance Imaging of Transplanted Porcine Neonatal Pancreatic Cell Clusters Labeled with Exendin-4-Conjugated Manganese Magnetism-Engineered Iron Oxide Nanoparticles.

Recently, we have shown that manganese magnetism-engineered iron oxide nanoparticles (MnMEIO NPs) conjugated with exendin-4 (Ex4) act as a contrast agent that directly trace implanted mouse islet ß-cells by magnetic resonance imaging (MRI). Here we further advanced this technology to track implanted porcine neonatal pancreatic cell clusters (NPCCs) containing ducts, endocrine, and exocrine cells. NPCCs from one-day-old neonatal pigs were isolated, cultured for three days, and then incubated overnight with MnMEIO-Ex4 NPs. Binding of NPCCs and MnMEIO-Ex4 NPs was confirmed with Prussian blue staining in vitro prior to the transplantation of 2000 MnMEIO-Ex4 NP-labeled NPCCs beneath the left renal capsule of six nondiabetic nude mice. The 7.0 T MRI on recipients revealed persistent hypointense areas at implantation sites for up to 54 days. The MR signal intensity of the graft on left kidney reduced 62-88% compared to the mirror areas on the contralateral kidney. Histological studies showed colocalization of insulin/iron and SOX9/iron staining in NPCC grafts, indicating that MnMEIO-Ex4 NPs were taken up by mature ß-cells and pancreatic progenitors. We conclude that MnMEIO-Ex4 NPs are excellent contrast agents for detecting and long-term monitoring implanted NPCCs by MRI.

Exendin-4-Conjugated Manganese Magnetism-Engineered Iron Oxide Nanoparticles as a Potential Magnetic Resonance Imaging Contrast Agent for Tracking Transplanted ß-Cells.

To specifically detect and trace transplanted islet ß-cells by magnetic resonance imaging (MRI), we conjugated manganese magnetism-engineered iron oxide nanoparticles (MnMEIO NPs) with exendin-4 (Ex4) which specifically binds glucagon-like peptide-1 receptors on the surface of ß-cells. The size distribution of MnMEIO and MnMEIO-Ex4 NPs were 67.8 ± 1.3 and 70.2 ± 2.3 nm and zeta potential 33.3 ± 0.5 and 0.6 ± 0.1 mV, respectively. MnMEIO and MnMEIO-Ex4 NPs with iron content ≤ 40 µg/mL did not affect MIN6 ß-cell viability and insulin secretion. Positive iron staining was found in MIN6 ß-cells loaded with MnMEIO-Ex4 NPs but not in those with MnMEIO NPs. A transmission electron microscope confirmed MnMEIO-Ex4 NPs were distributed in the cytoplasm of MIN6. In vitro MR images revealed a loss of signal intensity in MIN6 ß-cells labeled with MnMEIO-Ex4 NPs but not with MnMEIO NPs. After transplantation of islets labeled with MnMEIO-Ex4, the graft under kidney capsule could be visualized on MRI as persistent hypointense areas up to 17 weeks. Moreover, histology of the islet graft showed positive staining for insulin, glucagon and iron. Our results indicate MnMEIO-Ex4 NPs are safe and effective for the detection and long-term monitoring of transplanted ß-cells by MRI.

Effectiveness of a Problem-Solving Program in Improving Problem-Solving Ability and Glycemic Control for Diabetics with Hypoglycemia.

The purpose of this study was to evaluate the effects of a hypoglycemia problem-solving program (HPSP) on problem-solving ability and glycemic control in diabetics with hypoglycemia. This was a prospective, quasi-experimental study with two groups, using a pre- and post-repeated measures design. A total of 71 diabetic patients with hypoglycemia were purposively assigned to an experimental group (n = 34) and a control group (n = 37). The experimental group participated in an 8-week HPSP, and each weekly session lasted approximately 90 min, while the control group received usual care. Participants were assessed at baseline, 1, 3, and 6 months after intervention care. In the experimental group, 6 months after the HPSP intervention, HbA1c was superior to that before the intervention. In both groups, the score obtained using the hypoglycemia problem-solving scale (HPSS) was low before the intervention. In the experimental group, HPSS tracking improved at all stages after the intervention compared to before the intervention. In the control group, the HPSS score improved slightly in the first month and sixth months after usual care. There were significant differences between and within groups in HbA1c levels and HPSS score over time. The intervention based on the HPSP effectively improves HbA1c level and hypoglycemia problem-solving ability in patients with hypoglycemia.

Noninvasive Tracking of mPEG-poly(Ala) Hydrogel-Embedded MIN6 Cells after Subcutaneous Transplantation in Mice.

Recently, we demonstrated the feasibility of subcutaneous transplantation of MIN6 cells embedded in a scaffold with poly(ethylene glycol) methyl ether (mPEG)-poly(Ala) hydrogels. In this study, we further tracked these grafts using magnetic resonance (MR) and bioluminescence imaging. After being incubated overnight with chitosan-coated superparamagnetic iron oxide (CSPIO) nanoparticles and then mixed with mPEG-poly(Ala) hydrogels, MIN6 cells appeared as dark spots on MR scans. For in vivo experiments, we transfected MIN6 cells with luciferase and/or incubated them overnight with CSPIO overnight; 5 × 106 MIN6 cells embedded in mPEG-poly(Ala) hydrogels were transplanted into the subcutaneous space of each nude mouse. The graft of CSPIO-labeled MIN6 cells was visualized as a distinct hypointense area on MR images located at the implantation site before day 21. However, this area became hyperintense on MR scans for up to 64 days. In addition, positive bioluminescence images were also observed for up to 64 days after transplantation. The histology of removed grafts showed positive insulin and iron staining. These results indicate mPEG-poly(Ala) is a suitable scaffold for ß-cell encapsulation and transplantation. Moreover, MR and bioluminescence imaging are useful noninvasive tools for detecting and monitoring mPEG-poly(Ala) hydrogel-embedded MIN6 cells at a subcutaneous site.

Magnetic Resonance Imaging of Transplanted Porcine Neonatal Pancreatic Cell Clusters Labeled with Chitosan-Coated Superparamagnetic Iron Oxide Nanoparticles in Mice.

Neonatal pancreatic cell clusters (NPCCs) are potential tissues for the treatment of diabetes. Different from adult cells, they continuously proliferate and differentiate after transplantation. In this study, we utilized magnetic resonance imaging (MRI) to detect and monitor implanted NPCCs. NPCCs were isolated from one-day-old neonatal pigs, cultured for three days, and then incubated overnight with the contrast agent chitosan-coated superparamagnetic iron oxide (CSPIO) nanoparticles. In vitro, Prussian blue staining and MR scans of CSPIO-labeled NPCCs were performed. In vivo, we transplanted 2000 CSPIO-labeled NPCCs under the kidney capsule of nondiabetic nude mice. Recipients were scanned with 7.0T MRI. Grafts were removed for histology with insulin and Prussian blue staining. After being incubated overnight with CSPIO, NPCCs showed positive iron staining and appeared as dark spots on MR scans. After transplantation of CSPIO-labeled NPCCs, persistent hypointense areas were observed at recipients' implant sites for up to 54 days. Moreover, histology showed colocalization of the insulin and iron staining in 15-, 51- and 55-day NPCC grafts. Our results indicate that transplanted NPCCs survived and differentiated to ß cells after transplantation, and that MRI is a useful tool for the detection and monitoring of CSPIO-labeled NPCC grafts.

Implanted islet mass influences the effects of dipeptidyl peptidase-IV inhibitor LAF237 on transplantation outcomes in diabetic mice.

BACKGROUND: Previous studies showed inconsistent Results of the effects of dipeptidyl peptidase (DPP)-IV inhibitors on syngeneic mouse islet transplantation. We hypothesized that the implanted islet numbers are critical for the effects of DPP-IV inhibitors on the outcomes of transplantation. METHODS: One hundred and fifty or three hundred islets were syngeneically transplanted under the renal capsule of each streptozocin-diabetic C57BL/6 mouse and recipients were then treated without or with LAF237 (10 mg/kg/day, po) for 6 weeks. After transplantation, recipients' blood glucose, body weight and intraperitoneal glucose tolerance test (IPGTT) were followed-up periodically. The graft was removed for the measurement of ß-cell mass at 6 weeks. RESULTS: In recipients with 150 islets, it was not significantly different between the LAF237- treated group (n = 14) and control group (n = 14) in terms of the blood glucose, body weight, glucose tolerance at 2, 4 and 6 weeks or the graft ß-cell mass at 6 weeks. In contrast, in recipients with 300 islets, the LAF237-treated group (n = 24) did have a lower area under the curve of the IPGTT at 4 weeks (p = 0.0237) and 6 weeks (p = 0.0113) as well as more graft ß-cell mass at 6 weeks (0.655 ± 0.008 mg vs. 0.435 ± 0.006 mg, p = 0.0463) than controls (n = 24). CONCLUSIONS: Our findings revealed 6-week treatment of LAF237 improves glucose tolerance and increases graft ß-cell mass in diabetic mice transplanted with a sufficient number but not a marginal number of islets. These indicate that the effects of DPP-IV inhibitors are influenced by the implanted islet mass.

Codelivery of Sustainable Antimicrobial Agents and Platelet-Derived Growth Factor via Biodegradable Nanofibers for Repair of Diabetic Infectious Wounds.

More than half of diabetic wounds demonstrate clinical signs of infection at presentation and lead to poor outcomes. This work develops coaxial sheath-core nanofibrous poly(lactide-co-glycolide) (PLGA) scaffolds that are loaded with bioactive antibiotics and platelet-derived growth factor (PDGF) for the repair of diabetic infectious wounds. PDGF and PLGA/antibiotic solutions were pumped, respectively, into two independent capillary tubings for coaxial electrospinning to prepare biodegradable sheath-core nanofibers. Spun nanofibrous scaffolds sustainably released PDGF, vancomycin, and gentamicin for 3 weeks. The scaffolds also reduced the phosphatase and tensin homologue content, enhanced the amount of angiogenesis marker (CD31) around the wound area, and accelerated healing in the early stage of infected diabetic wound repair. Antibiotic/biomolecule-loaded PLGA nanofibers may provide a very effective way to aid tissue regeneration at the sites of infected diabetic wounds.

Enhancement of Subcutaneously Transplanted ß Cell Survival Using 3D Stem Cell Spheroids with Proangiogenic and Prosurvival Potential.

Islet transplantation has been demonstrated to be a promising therapy for type 1 diabetes mellitus. Although it is a minimally invasive operating procedure and provides easy access for graft monitoring, subcutaneous transplantation of the islet only has limited therapeutic outcomes, owing to the poor capacity of skin tissue to foster revascularization in a short period. Herein, 3D cell spheroids of clinically accessible umbilical cord blood mesenchymal stem cells and human umbilical vein endothelial cells are formed and employed for codelivery with ß cells subcutaneously. The 3D stem cell spheroids, which can secrete multiple proangiogenic and prosurvival growth factors, induce robust angiogenesis and prevent ß cell graft death, as indicated by the results of in vivo bioluminescent tracking and histological analysis. These experimental data highlight the efficacy of the 3D stem cell spheroids that are fabricated using translationally applicable cell types in promoting the survival and function of subcutaneously transplanted ß cells.

Core-shell insulin-loaded nanofibrous scaffolds for repairing diabetic wounds.

Patients with diabetes mellitus have up to a 15% lifetime risk of non-healing and poorly healing wounds. This work develops core-shell nanofibrous bioactive insulin-loaded poly-D-L-lactide-glycolide (PLGA) scaffolds that release insulin in a sustained manner for repairing wounds in diabetic rats. To prepare the biodegradable core-shell nanofibers, PLGA and insulin solutions were fed into two capillary tubes of different sizes that were coaxially electrospun using two independent pumps. The scaffolds sustainably released insulin for four weeks. The hydrophilicity and water-containing capacity of core-shell nanofibrous insulin/PLGA scaffolds significantly exceeded those of blended nanofibrous scaffolds. The nanofibrous core-shell insulin-loaded scaffold reduced the amount of type I collagen in vitro, increased the transforming growth factor-beta content in vivo, and promoted diabetic would repair. The core-shell insulin-loaded nanofibrous scaffolds prolong the release of insulin and promote diabetic wound healing.

Nanofibrous vildagliptin-eluting stents enhance re-endothelialization and reduce neointimal formation in diabetes: in vitro and in vivo.

BACKGROUND: The high lifetime risk of vascular disease is one of the important issues that plague patients with diabetes mellitus. Systemic oral vildagliptin administration favors endothelial recovery and inhibits smooth muscle cell (SMC) proliferation. However, the localized release of vildagliptin in the diabetic vessel damage has seldom been investigated. RESEARCH DESIGN AND METHODS: In this work, nanofiber-eluting stents that loaded with vildagliptin, a dipeptidyl peptidase-4 enzyme (DPP-4) inhibitor, was fabricated to treat diabetic vascular disease. To prepare nanofibers, the poly (D,L)-lactide-co-glycolide (PLGA) and vildagliptin were mixed using hexafluoroisopropanol and electrospinning process. In vitro and in vivo release rates of the vildagliptin were characterized using high-performance liquid chromatography. RESULTS: Effective vildagliptin concentrations were delivered for more than 28 days from the nanofibrous membranes coating on the surface of the stents in vitro and in vivo. The vildagliptin-eluting PLGA membranes greatly accelerated the recovery of diabetic endothelia and reduced SMC hyperplasia. The type I collagen content of the diabetic vascular intimal area that was treated by vildagliptin-eluting stents was lower than that of the non-vildagliptin-eluting group. CONCLUSION: The experimental results revealed that stenting with vildagliptin-eluting PLGA membranes could potentially promote healing for diabetic arterial diseases.

Promoting vascular healing using nanofibrous ticagrelor-eluting stents.

OBJECTIVE: The current treatment of atherosclerotic coronary heart disease with limus-eluting stents can lead to incomplete endothelialization and substantial impairment of arterial healing relative to treatment with bare-metal stents. The sustained and local delivery of ticagrelor, a reversibly binding P2Y12 receptor inhibitor, using hybrid biodegradable nanofibers/stents, was developed to reduce neointimal formation and endothelial dysfunction. METHODS: In this investigation, a solution of ticagrelor, poly(D,L)-lactide-co-glycolide, and hexafluoro isopropanol was electrospun to fabricate ticagrelor-eluting nanofibrous drug-eluting stents. The in vitro and in vivo ticagrelor concentrations were measured using a high-performance liquid chromatography assay. The effectiveness of ticagrelor-eluting stents was examined relative to that of sirolimus-eluting stents. RESULTS: Adequate ticagrelor levels were detected for four weeks in vitro. Less HES5-positive labeling was found near the ticagrelor-eluting stented vessels (0.33±0.12) than close to the sirolimus-eluting stented vessels (0.57±0.15) (p<0.05). Four weeks after deployment, the ticagrelor-eluting stent also exhibited an up-regulated local expression of SOD1 in the stenting area (p<0.001). The ticagrelor-eluting stent substantially preserved endothelial function and re-endothelialization, minimized inflammatory responses, and inhibited neointimal hyperplasia. CONCLUSION: Ticagrelor-eluting stents may provide an alternative route for treating patients at a high risk of bleeding to preserve endothelial recovery and to reduce smooth muscle proliferation.

Oral Nonviral Gene Delivery for Chronic Protein Replacement Therapy.

Efficient nonviral oral gene delivery offers an attractive modality for chronic protein replacement therapy. Herein, the oral delivery of insulin gene is reported by a nonviral vector comprising a copolymer with a high degree of substitution of branched polyethylenimine on chitosan (CS-g-bPEI). Protecting the plasmid from gastric acidic degradation and facilitating transport across the gut epithelium, the CS-g-bPEI/insulin plasmid DNA nanoparticles (NPs) can achieve systemic transgene expression for days. A single dose of orally administered NPs (600 µg plasmid insulin (pINS)) to diabetic mice can protect the animals from hyperglycemia for more than 10 d. Three repeated administrations spaced over a 10 d interval produce similar glucose-lowering results with no hepatotoxicity detected. Positron-emission-tomography and computed-tomography images also confirm the glucose utilization by muscle cells. While this work suggests the feasibility of basal therapy for diabetes mellitus, its significance lies in the demonstration of a nonviral oral gene delivery system that can impact chronic protein replacement therapy and DNA vaccination.

An Intestinal "Transformers"-like Nanocarrier System for Enhancing the Oral Bioavailability of Poorly Water-Soluble Drugs.

Increasing the intestinal dissolution of orally administered poorly water-soluble drugs that have poor oral bioavailability to a therapeutically effective level has long been an elusive goal. In this work, an approach that can greatly enhance the oral bioavailability of a poorly water-soluble drug such as curcumin (CUR) is developed, using a "Transformers"-like nanocarrier system (TLNS) that can self-emulsify the drug molecules in the intestinal lumen to form nanoemulsions. Owing to its known anti-inflammation activity, the use of CUR in treating pancreatitis is evaluated herein. Structural changes of the TLNS in the intestinal environment to form the CUR-laden nanoemulsions are confirmed in vitro. The therapeutic efficacy of this TLNS is evaluated in rats with experimentally induced acute pancreatitis (AP). Notably, the CUR-laden nanoemulsions that are obtained using the proposed TLNS can passively target intestinal M cells, in which they are transcytosed and then transported into the pancreatic tissues via the intestinal lymphatic system. The pancreases in rats that are treated with the TLNS yield approximately 12 times stronger CUR signals than their counterparts receiving free CUR, potentially improving the recovery of AP. These findings demonstrate that the proposed TLNS can markedly increase the intestinal drug dissolution, making oral delivery a favorable noninvasive means of administering poorly water-soluble drugs.

Porcine Neonatal Pancreatic Cell Clusters Maintain Their Multipotency in Culture and After Transplantation.

Ductal epithelium is primarily detected in porcine neonatal pancreatic cell clusters (NPCCs) bearing grafts, suggesting that transplants might exhibit progenitor-like phenotypes. Here we found that soon after NPCC isolation, PDX1+/insulin- and SOX9+ pancreatic progenitor-like cells dramatically increased while dual-hormonal progenitor-like cells were routinely observed in NPCC culture. After transplantation (Tx), insulin+ cells increased and PDX1+ and SOX9+ cells gradually decreased in both non-diabetic (NDM) and streptozotocin-induced diabetic (DM) grafts over 2 months. Strikingly, a significantly higher percentage of insulin+ cells were detected in 9-day and 16-day, but not in 23-day, 30-day and 60-day grafts implying that hyperglycemia could only facilitate NPCC-derived ß cells early post-Tx. A higher percentage of NPCC-derived ß cells in early DM grafts was determined via an enhanced neogenic differentiation based on the detection of insulin+ cells budding out from PDX1+/SOX9+ epithelium. Interestingly, a drop in SOX9+ progenitor-like cells was detected 16 days post-Tx in DM grafts whilst PDX1+ cells do not show a significant difference until 60 days post-Tx between DM and NDM grafts, demonstrating that distinct progenitor-like populations fuel new ß cells post-Tx. In conclusion, PDX1+/SOX9+ cells could be quickly activated after NPCC isolation, maintain their multipotency in culture and differentiate into new ß cell post-Tx.

Factors Affecting the Ability of People With Diabetes to Avoid Hypoglycemia.

BACKGROUND: Hypoglycemia is recognized as a limiting factor in diabetes management. Fear of experiencing hypoglycemia may lead to lower quality of life, impaired glycemic control, and emotional distress, all of which impair the ability of patients to self-manage their diabetes effectively. Problem solving is central to diabetes self-management and may help patients achieve effective self-care of their disease. PURPOSE: The aim of this study was to investigate the ability of people with diabetes to avoid hypoglycemia and to explore associated factors. METHODS: A cross-sectional, descriptive design was used for the study. Data were collected using a demographic and disease characteristics datasheet, the Hypoglycemic Problem Solving Scale, and the Disease-Associated Negative Mood Scale. RESULTS: Three hundred thirteen participants were recruited, with a mean age of 55.49 years. The average item score for the questions on hypoglycemic problem-solving ability was 2.43 (SD = 0.75). In comparing Hypoglycemic Problem Solving Scale subscales, participants scored highest on the problem orientation subscales and lowest on the problem-solving skills subscales. Multiple regression analysis revealed that being younger and unmarried and having a higher level of education, a diagnosis of Type 1 diabetes, and a lower negative mood score were each significantly associated with greater problem-solving ability as regards hypoglycemic events. CONCLUSIONS/IMPLICATIONS FOR PRACTICE: We suggest that patients with diabetes, especially those who are older or with lower levels of education, receive disease-related psychological interventions and that healthcare professionals teach problem-solving abilities in conjunction with hypoglycemia management.

Safety and efficacy of self-assembling bubble carriers stabilized with sodium dodecyl sulfate for oral delivery of therapeutic proteins.

Sodium dodecyl sulfate (SDS) is generally regarded as a potent permeability enhancer in oral formulations; however, one concern related to the use of any permeation enhancer is its possible absorption of unwanted toxins during the period of epithelial permeability enhancement. In this work, the safety and efficacy of an SDS-containing bubble carrier system that is developed from an orally administered enteric-coated capsule are evaluated. The bubble carriers comprise diethylene triamine pentaacetic acid (DTPA) dianhydride, sodium bicarbonate (SBC), SDS, and insulin. Upon exposure to the intestinal fluid, DTPA dianhydride hydrolyzes to yield acids, and SBC rapidly reacts with these acids to generate CO2, producing bubble carriers, each containing a self-assembling water film. The hydrophilic insulin is entrapped in the self-assembled water film, which is stabilized by SDS. The SDS in the bubble carrier system can act as a dissolution enhancer in the dispersion of insulin molecules, as a surfactant that stabilizes the bubble carriers, as a protease inhibitor that protects the protein drug, and as a permeation enhancer that augments its oral bioavailability. Hence, a significant increase in the plasma insulin level and an excellent blood glucose-lowering response in diabetic rats are effectively achieved. Moreover, the enhancement of epithelial permeation by this SDS-containing formulation does not promote the absorption of intestinal endotoxins. The above facts indicate that the bubble carrier system that is stabilized by SDS can be used as a safe and potent carrier in the oral delivery of therapeutic proteins.