Using Ultrasound-targeted Microbubble Destruction Research Articles

MSCs biomimetic ultrasonic phase change nanoparticles promotes cardiac functional recovery after acute myocardial infarction

Acute Myocardial Infarction (AMI) has seen rising cases, particularly in younger people, leading to public health concerns. Standard treatments, like coronary artery recanalization, often don't fully repair the heart's microvasculature, risking heart failure. Advances show that Mesenchymal Stromal Cells (MSCs) transplantation improves cardiac function after AMI, but the harsh microenvironment post-AMI impacts cell survival and therapeutic results. MSCs aid heart repair via their membrane proteins and paracrine extracellular vesicles that carry microRNA-125b, which regulates multiple targets, preventing cardiomyocyte death, limiting fibroblast growth, and combating myocardial remodeling after AMI. This study introduces ultrasound-responsive phase-change bionic nanoparticles, leveraging MSCs' natural properties. These particles contain MSC membrane and microRNA-125b, with added macrophage membrane for stability. Using Ultrasound Targeted Microbubble Destruction (UTMD), this method targets the delivery of MSC membrane proteins and microRNA-125b to AMI's inflamed areas. This aims to enhance cardiac function recovery and provide precise, targeted AMI therapy.

Role of Ultrasonic Microbubbles in Treating Rheumatoid Arthritis: Enhancing the Efficacy of Tocilizumab via Contrast-Enhanced Ultrasound-Monitored, Ultrasound-Targeted Microbubble Destruction.

Our aim was to explore the feasibility of using ultrasound-targeted microbubble destruction (UTMD) to deliver tocilizumab and enhance its efficacy in treating rheumatoid arthritis (RA). Rats with adjuvant-induced arthritis were randomly assigned to one of five treatment groups: group 1, tocilizumab+microbubbles (MBs)+UTMD; group 2, tocilizumab+MBs; group 3, tocilizumab+saline; group 4, MBs+UTMD; group 5, no treatment. We employed a commercially available ultrasound (US) machine capable of performing contrast-enhanced ultrasound (CEUS) and UTMD simultaneously using a single probe. CEUS was performed to monitor the entry and collapse of MBs. After treatment, the rats' left hindlimb paws were harvested for immunohistochemical staining of interleukin-6 (IL-6) and tumor necrosis factor α (TNF-α). After injection of the mixture of drugs and MBs with UTMD, significant enhancement was seen in the inflamed hindlimb paw regions, which subsided immediately on exposure to low-frequency US beams and re-appeared in the intervals between beam exposures. IL-6 expression was significantly lower in groups 1, 2 and 3 than in groups 4 and 5 (p < 0.01). Group 1 had the lowest level of IL-6 expression (p [G1 vs. G2] < 0.01, p [G1 vs. G3] < 0.01). The levels of TNF-α expression in groups 1, 2, and 3 were significantly lower than those in groups 4 and 5, but no difference was observed in these levels between groups 1-3. UTMD shows promise in enhancing the treatment efficacy of anti-IL-6 drugs for RA treatment.

Prevention of doxorubicin induced cardiomyopathy after delivery of modified acidic fibroblast growth factor by using ultrasound-targeted microbubble destruction: a preliminary experimental study

Objective To observe the preventive effects of doxorubicin-induced cardiomyopathy (DOX-CM) rats after delivery of modified acidic fibroblast growth factor (MaFGF) by using ultrasound-targeted microbubble destruction (UTMD). Methods Fifty male SD rats were randomly divided into five groups: ①Control group; ②DOX-CM group; ③MaFGF group; ④UTMD group; ⑤MaFGF+ UTMD group. ②~⑤groups were intraperitoneally injected with doxorubicin to induce DOX-CM and given the corresponding interventions. Six weeks after intervention, all rats were underwent conventional echocardiography and velocity vector imaging (VVI) exams. Left ventricular internal dimension-diastole (LVIDd), left ventricular internal dimension-systole (LVIDs), left ventricular ejection fraction (LVEF) and left ventricular fractional shortening (LVFS) were measured using conventional echocardiography. The segmental mean peak systolic radial velocity (Vs), systolic radial strain (Sr), and systolic radial strain rate (SRr) of six walls at the papillary muscle level were measured in left ventricular short-axis view by VVI. At last, myocardial tissues of all rats were stained with HE to observe the myocardial tissue morphology and with Van Gieson (VG) to calculate the collagen volume fractions (CVF). Results Six weeks after intervention, heart weight/body weight (HW/BW), LVIDs and LVIDd of MaFGF+ UTMD group were significantly decreased compared with those of DOX-CM group(P<0.05), while the LVEF, LVFS, Vs, Sc and Sr were significantly increased (P<0.05). Van Gieson stains showed that the collagen volume fractions (CVF) of DOX-CM groups significantly increased compared with that of the control group (P<0.01), while the CVF of MaFGF+ UTMD groups significantly decreased (P<0.01). Conclusions Delivery of MaFGF to myocardium in DOX-CM rats by UTMD could retard the formation of myocardial fibrosis and effectively protect the left ventricular function of DOX-CM rats. Key words: Echocardiography; Microbubbles; Acidic fibroblast growth factor; Cardiomyopathy; Doxorubicin

Targeted myocardial delivery of GDF11 gene rejuvenates the aged mouse heart and enhances myocardial regeneration after ischemia-reperfusion injury.

Ischemic cardiac injury is the main contributor to heart failure, and the regenerative capacity of intrinsic stem cells plays an important role in tissue repair after injury. However, stem cells in aged individuals have reduced regenerative potential and aged tissues lack the capacity to renew. Growth differentiation factor 11 (GDF11), from the activin-transforming growth factor β superfamily, has been shown to promote stem cell activity and rejuvenation. We carried out non-invasive targeted delivery of the GDF11 gene to the heart using ultrasound-targeted microbubble destruction (UTMD) and cationic microbubble (CMB) to investigate the ability of GDF11 to rejuvenate the aged heart and improve tissue regeneration after injury. Young (3 months) and old (21 months) mice were used to evaluate the expression of GDF11 mRNA in the myocardium at baseline and after ischemia/reperfusion (I/R) and myocardial infarction. GDF11 expression decreased with age and following myocardial injury. UTMD-mediated delivery of the GDF11 plasmid to the aged heart after I/R injury effectively and selectively increased GDF11 expression in the heart, and improved cardiac function and reduced infarct size. Over-expression of GDF11 decreased senescence markers, p16 and p53, as well as the number of p16+ cells in old mouse hearts. Furthermore, increased proliferation of cardiac stem cell antigen 1 (Sca-1+) cells and increased homing of endothelial progenitor cells and angiogenesis in old ischemic hearts occurred after GDF11 over-expression. Repetitive targeted delivery of the GDF11 gene via UTMD can rejuvenate the aged mouse heart and protect it from I/R injury.

Spatial Control of Gene Expression by Nanocarriers Using Heparin Masking and Ultrasound-Targeted Microbubble Destruction.

We developed a method to spatially control gene expression following nonviral delivery of DNA. This method includes surface-modifying DNA nanocarriers with heparin to inhibit passive gene transfer in both the target and the off-target tissues and using ultrasound-targeted microbubble destruction (UTMD) to selectively activate heparin-inhibited gene transfer at the target site. We observed that the engraftment of heparin onto the surface of cationic liposomes reduced off-target gene expression in the liver, a major site of nanoplex accumulation, by more than 700-fold compared to the nonheparinized PEGylated liposomes. We further observed that tumor-directed UTMD increased gene transfer with heparin-modified nanoplexes by more than 10-fold. This method augmented tumor-to-liver selectivity of gene expression by 4000-fold compared to controls. We conclude that heparinization of DNA nanocarriers in conjunction with localized activation of gene transfer by UTMD may enable greater spatial control over genetic therapy.

Abstract 2919: Ultrasound-mediated neuropilin-1 shRNA minicircle delivery inhibits tumour growth in an orthotopic human pancreatic adenocarcinoma model

Abstract Background: Pancreatic ductal adenocarcinoma (PDAC) is characterized by an intense fibrotic reaction termed tumour desmoplasia/fibrosis, which is partially responsible for its aggressive nature. The pro-fibrotic cytokine TGFβ1 promotes fibroblast accumulation in several pathological disorders, including cancer via endothelial-to-mesenchymal transition (EndMT), thereby implicating EndMT as a potential therapeutic target. Previous studies have demonstrated angiogenic co-receptor, neuropilin-1 (NRP-1) as a co-receptor for TGFβ1 in mediating several oncogenic processes. NRP-1 expression and TGFβ1 signaling have been shown to be up-regulated in PDAC. However, the therapeutic benefits of targeting NRP-1 in PDAC remain unexplored. We therefore hypothesized that targeting NRP-1 in PDAC may inhibit TGFβ1-mediated EndMT, tumour perfusion and tumour growth. Methods: Orthotopic tumours were generated in RNU athymic rats from BxPC-3 human pancreatic cancer cells using the implantation technique. Animals were delivered at day 28 post surgery with either empty minicircle-DNA (150 μg) or shNRP-1 minicircle-DNA (200 μg) using ultrasound targeted microbubble destruction (UTMD). For UTMD, ultrasound was transmitted using a phased array transducer (S3, Sonos 5500) at 1.3 MHz and a transmit power of 0.9 W (120 V, 9 mA), and at a pulsing interval of 5 s. Contrast enhanced ultrasound perfusion imaging was performed on day 28 (gene delivery) and day 56 (end-point) with standardized analyses. Tumour tissues and remote organs were collected for evaluation of tumour volume and other downstream analyses like real-time polymerase chain reaction (RT-PCR), immunoblotting, immunohistochemistry and Masson's trichrome staining (for fibrosis). Results: RT-PCR and immunoblotting demonstrated successful silencing of NRP-1 in shNRP-1 minicircle group. Immunohistochemistry demonstrated a typical ductal morphology and substantial collagen content within the tumours. Histological assessments further revealed reduced NRP-1 expression and reduced fibrosis in shNRP-1 minicircle group. Significant changes were observed in EndMT markers at transcript level and protein level in shNRP-1 minicircle delivered animals compared to empty minicircle. At day 56, profound reductions were observed in tumour volumes in shNRP-1 minicircle delivered animals. Tumour area and perfusion were found to be greater and more complete throughout the tumour in empty minicircle group, while reduced area and perfusion was observed in shNRP-1 minicircle group. At day 56, tumour microvascular blood flow and blood volume were markedly reduced in the shNRP-1 minicircle group. Conclusions: Overall, these in vivo results suggest that loss of NRP-1 leads to reduced fibrosis and tumour growth in PDAC possibly through limiting EndMT and NRP-1 mediated angiogenesis, making NRP-1 as a potential therapeutic target against PDAC. Citation Format: Pratiek N. Matkar, Krishna K. Singh, Gerald J. Prud’homme, David W. Hedley, Howard Leong-Poi. Ultrasound-mediated neuropilin-1 shRNA minicircle delivery inhibits tumour growth in an orthotopic human pancreatic adenocarcinoma model. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2919.

Improvement of left ventricular myocardial perfusion after acidic fibroblast growth factor delivered by using ultrasound-targeted microbubble destruction in rats with diabetic cardiomyopathy: an preliminary study

Objective To investigate the protective effects of left ventricular myocardial perfusion after delivery of acidic fibroblast growth factor (aFGF) in rats with diabetic cardiomyopathy (DCM) by using ultrasound-targeted microbubble destruction (UTMD) with real-time myocardial contrast echocardiography(RT-MCE). Methods Among 64 male SD rats, 52 rats were randomly selected and were induced DCM by streptozotocin through intraperitoneal injecting, the other rats as normal control group. DCM rats were randomly divided into the DCM model group, aFGF only group, SonoVue-aFGF group and the SonoVue-aFGF+ UTMD group in this study. The aFGF only group rats were injected with aFGF solution through tail vein, the SonoVue-aFGF group were injected with SonoVue-aFGF solution through tail vein and SonoVue-aFGF+ UTMD group rats were injected with SonoVue-aFGF solution through tail vein and using UTMD simultaneously. All rats underwent conventional echocardiography and RT-MCE exams before and 4 weeks after intervention. Left ventricular ejection fraction (LVEF) and left ventricular fraction shortening(LVFS) were measured by conventional echocardiography. The plateau intensity (A), initial slope of the curve (β) and myocardial blood flow (A×β) of left ventricular anterior wall at the papillary muscle level were measured in left ventricular short-axis view by RT-MCE. Results Before intervention, LVEF and LVFS in the DCM model group, aFGF only group, SonoVue-aFGF group and the SonoVue-aFGF+ UTMD group were significantly lower than in the normal control group (P 0.05), however A and A×β in the SonoVue-aFGF+ UTMD group were significantly increased than those in the DCM model group(P<0.01). Compared with the same group before intervention, A and A×β in the SonoVue-aFGF+ UTMD group were higher (P<0.05) and these in the DCM model group were lower during four weeks after intervention (P<0.05). Conclusions Acidic fibroblast growth factor delivered by using UTMD can improve the left ventricular myocardial perfusion in diabetic cardiomyopathy rats. Key words: Echocardiography; Diabetic cardiomyopathies; Fibroblast growth factor; Microbubbles; Ventricular function, left

Use of ultrasound-targeted microbubble destruction to transfect IGF-1 cDNA to enhance the regeneration of rat wounded Achilles tendon in vivo.

The purpose of this study was to determine whether using ultrasound-targeted microbubble destruction (UTMD) to transfect rat wounded Achilles tendon with insulin-like growth factor-1 (IGF-1) cDNA would enhance tissue regeneration. Forty rats with injured Achilles tendons were transfected with IGF-1 cDNA and divided into: (1) control group, (2) plasmid-only group, (3) plasmid+ultrasound group and (4) plasmid+microbubbles+ultrasound group. The IGF-1 cDNA expression of the Achilles tendons was evaluated by histological adhesion finding, quantitative real-time reverse transcription PCR examination and biomechanical test. The adhesion scores in group 4 were lowest at weeks 2 and 8 (P<0.05). The IGF-1 expression in the Achilles tendons was highest in group 4 at weeks 2 and 8 (P<0.05). Compared with those of other three groups, the granulation tissues and inflammatory-cell infiltration were lighter in group 4 at week 2, and the scars on the tendons in group 4 were less evident at week 8. The messenger RNA (mRNA) of IGF-1 of group 4 was upregulated at weeks 2 and 8 (P<0.01). Groups 4 and 3 showed a greater maximum load, stiffness and ultimate stress (P<0.05). Maximum load, stiffness and ultimate stress of healing Achilles tendons in group 4 were highest at weeks 2 and 8 (P<0.05).

Transfection of wtp53 and Rb94 Genes Into Retinoblastomas of Nude Mice by Ultrasound-Targeted Microbubble Destruction

Using ultrasound-targeted microbubble destruction (UTMD), we transfected both wild-type p53 (wtp53) and Rb94 genes into retinoblastomas (RBs) of nude mice to investigate the inhibitory role of these two genes in RB development. The 40 tumor-bearing mice, which had been established by sub-retinal injection of an HXO-Rb44 cell suspension, were randomly divided into five groups: blank control group (C); blank plasmid group (M); wtp53 plasmid group (p53); Rb94 plasmid group (Rb94); wtp53 + Rb94 plasmid group (p53 + Rb94). For preparation of the DNA-loaded microbubbles, a pre-determined amount of blank plasmid, pVIVO1-p53, pVIVO1-Rb94 or pVIVO1-p53-Rb94 was added and mixed with the microbubbles. Then, these DNA-loaded microbubbles were respectively transfected into the animal model by UTMD. Vascular endothelial growth factor level and microvessel density of the tumor were determined by immunohistochemical staining. Apoptosis of tissues was detected by terminal deoxynucleotidyl transferase dUTP nick end labeling staining. Expression of wtp53 and Rb94 at both the gene and protein levels was detected by RT-PCR (reverse transcription polymerase chain reaction) and Western blot, respectively. Transfection of both genes had greater inhibitory effects on RB development and resulted in lower levels of vascular endothelial growth factor, lower microvessel density and more obvious apoptosis than single-gene transfection (p < 0.05). The results indicate that the transfection of both genes into the RB by UTMD more strongly inhibited RB growth than transfection of a single gene.

Successful β cells islet regeneration in streptozotocin-induced diabetic baboons using ultrasound-targeted microbubble gene therapy with cyclinD2/CDK4/GLP1

Both major forms of diabetes mellitus (DM) involve β-cell destruction and dysfunction. New treatment strategies have focused on replenishing the deficiency of β-cell mass common to both major forms of diabetes by islet transplantation or β-cell regeneration. The pancreas, not the liver, is the ideal organ for islet regeneration, because it is the natural milieu for islets. Since islet mass is known to increase during obesity and pregnancy, the concept of stimulating pancreatic islet regeneration in vivo is both rational and physiologic. This paper proposes a novel approach in which non-viral gene therapy is targeted to pancreatic islets using ultrasound targeted microbubble destruction (UTMD) in a non-human primate model (NHP), the baboon. Treated baboons received a gene cocktail comprised of cyclinD2, CDK, and GLP1, which in rats results in robust and durable islet regeneration with normalization of blood glucose, insulin, and C-peptide levels. We were able to generate important preliminary data indicating that gene therapy by UTMD can achieve in vivo normalization of the intravenous (IV) glucose tolerance test (IVGTT) curves in STZ hyperglycemic-induced conscious tethered baboons. Immunohistochemistry clearly demonstrated evidence of islet regeneration and restoration of β-cell mass.

Improvement of left ventricular systolic function after acidic fibroblast growth factor delivered by using ultrasound-targeted microbubble destruction in diabetic cardiomyopathy in rats:preliminary experimental study

Objective To investigate the protective effects of left ventricular systolic function after delivery of acidic fibroblast growth factor (aFGF) in diabetic cardiomyopathy(DCM) in rats by using ultrasound-targeted microbubble destruction (UTMD) with velocity vector imaging(VVI).Methods The sixty-four male SD rats comprised fifty-two DCM rats,which were induced by streptozotocin through intraperitoneal injecting,and twelve normal rats as normal control group in the study.The DCM rats were randomly divided into four groups and processed as follows:DCM model group,aFGF only group,SonoVueaFGF group,SonoVue-aFGF + UTMD group.Before and four weeks after intervention,all rats underwent M-mode echocardiography and VVI exams.Left ventricular ejection fraction (LVEF),left ventricular short axis shorten rate (LVFS) were measured using M-mode echocardiography.The mean peak circumferential strain (Sc) and radial strain (Sr) of six wall at the papillary muscle level were measured in left ventricular short-axis view by VVI.At last,myocardial tissue of all rats were stained with CD31 immunohistochemistry to quantify myocardial microvascular density(MVD).Results Before intervention,LVFS,LVEF,Sc and Sr in each experimental group were significantly lower than in the normal control group (P ＜0.01).Four weeks after intervention,Sc,Sr and MVD in the SonoVue-aFGF + UTMD group were significantly increased than in the DCM model group (P ＜0.01),and the above indexes slightly increased in both aFGF only group and SonoVue-aFGF group than those of DCM model group (P ＞0.05).Compared with before intervention,Sc and Sr in the SonoVue-aFGF + UTMD group were significantly higher and these in the DCM model group were significantly lower (P ＜0.05-0.01),in the aFGF only group and SonoVue-aFGF group were slightly increased(P ＞0.05).Conclusions Delivery of aFGF to myocardium in DCM rats by UTMD could enhance angiogenesis and improve the left ventricular systolic function. Key words: Echocardiography; Microbubbles; Ventricular function, left ; Fibroblast growth factor

GW24-e3129 Protective Effects and Possible Mechanism on Left Ventricular Function after Delivery of Acidic Fibroblast Growth Factor to Diabetic Myocardium by Using Ultrasound-targeted Microbubble Destruction

ObjectivesTo observe the protective effects of left ventricular function in diabetic cardiomyopathy rats after delivery of acidic fibroblast growth factor (aFGF) to myocardium by using ultrasound-targeted microbubble destruction (UTMD) and...

Experimental Research of RB94 Gene Transfection Into Retinoblastoma Cells Using Ultrasound-Targeted Microbubble Destruction

The purpose of this study was to explore the transfection of the recombinant expression plasmid pEGFP-C1/RB94 into human retinoblastoma cells (HXO-Rb44) using ultrasound-targeted microbubble destruction (UTMD). pEGFP-C1/RB94 was transfected into HXO-Rb44 in vitro by UTMD, with liposome as the positive control. After 24 to 72 h, the expression of the reporter gene enhanced green fluorescent protein (EGFP) was observed using fluorescent microscopy and flow cytometry. The cell viability of HXO-Rb44 was measured by a MTT assay. The mRNA and proteins of RB94, caspase-3 and Bax were analyzed by reverse transcription polymerase chain reaction (RT-PCR) and Western blot. Moreover, the apoptosis rate and cell cycle progression of the cells were detected by flow cytometry. This study demonstrated that UTMD can enhance the transfection efficiency of RB94, which has an obvious impact on the inhibition of the growth process of retinoblastoma cells, suggesting that the combination of UTMD and RB94 compounds might be a useful tool for use in the gene therapy of retinoblastoma.

Ultrasound contrast agent-mediated HSV1-TK/GCV suicide gene system for hepatocellular carcinoma in vitro

Objective To explore the specific lethal effectiveness of treating hepatoma carcinoma in vitro using ultrasound-targeted microbubble (UM) destruction mediated by HSV1-TK/GCV suicide gene system,and indentify the effects of UM destruction treatment on the cell viability.Methods HepG2 cells were seeded in the 24-well plates,and randomly divided into 5 groups:(A) blank control group; (B)HSV1-TK:pIRES2-EGFP-TK only; (C) HSV1-TK + UM:gene and ultrasound contrast agent; (D)HSV1-TK + US:gene and UM + pulsed-ultrasound; (E) HSV1-TK + UM + US:gene + US + pIRES2-EGFP-TK + pulsed-ultrasound.After 24 h,the expression of EGFP was observed under the fluorescent microscopy and quantified by flow cytometry.Western Blotting was used to detect the expression of TK protein.Twenty-four h after transfection,different concentrations of GCV (0,0.1,1.0,10.0,50.0,100.0,500.0,1000.0 mg/L) were added into the culture medium,then cells were cultured for another 72 h.The viability of HepG2 cells was measured by methyl thiazol tetrazolium (MTT) assay.Results Gene fluorescence intensity and tansfection efficiency in group E were higher than other groups.The gene transfection efficiency in groups A,B,C,D and E was 0％,0.39％,0.61％,1.10％ and 36.00％ respectively.TK protein in group E was higher than other groups ( P ＜ 0.05 ).The cell viability had no significant difference among group before addition of GCV.Almost all HepG2 cells in group E were killed when the concentration of GCV was above 50 mg/L,but almost no killing effect was observed in other groups after addition of different concentrations of GCV.Conclusion Ultrasound-targeted microbubble destruction can enhance the efficiency of gene transfection without obvious damage to cell viability in HepG2 cells.The method can also enhance the killing effect of HSV1-TK/GCV suicide gene system in vitro. Key words: Carcinoma,hepatocellular; Ultrasound; Contrast agent; HSV1-TK/GCV; Gene therapy

PHD2-shRNA interference by ultrasound targeted microbubble destruction facilitates angiogenesis and enhances myocardial function in ischemic/reperfusion injury in rats

ObjectiveHypoxia-inducible factor-1α (HIF-1α), a transcription factor, is naturally degraded by prolyl hydroxylase-2 (PHD2). During ischemic and reperfusion injury, upregulation of HIF-1α activates downstream angiogenic genes. We hypothesise inhibition of HIF-1α...

Regeneration of pancreatic islets in vivo by ultrasound-targeted gene therapy

The present study uses a novel approach to gene therapy in which plasmid DNA is targeted to the pancreas in vivo using ultrasound targeted microbubble destruction (UTMD) to achieve islet regeneration. Intravenous microbubbles carrying plasmids are destroyed within the pancreatic microcirculation by ultrasound, achieving local gene expression that is further targeted to beta-cells by a modified rat insulin promoter (RIP3.1). A series of genes implicated in endocrine development were delivered to rats 2 days after streptozotocin-induced diabetes. The genes PAX4, Nkx2.2, Nkx6.1, Ngn3, and Mafa produced alpha cell hyperplasia, but no significant improvement in beta cell mass or blood glucose 30 days after UTMD. In contrast, RIP3.1-NeuroD1 promoted islet regeneration from surviving beta-cells, with normalization of glucose, insulin, and C-peptide at 30 days. In a longer-term experiment, 4 of 6 rats had return of diabetes at 90 days, accompanied by beta cell apoptosis on Tunel staining. Pre-treatment with the JNK inhibitor SP600125 successfully blocked beta-cell apoptosis and resulted in restoration of beta cell mass and normalization of blood glucose for up to 90 days. This technique allows in vivo islet regeneration, restoration of beta cell mass, and normalization of blood sugar, insulin, and C-peptide in rats without viruses.