The mitochondria have emerged as a novel target for cancer chemotherapy primarily due to their central roles in energy metabolism and apoptosis regulation. Here, we report a new molecular approach to achieve high levels of tumor- and mitochondria-selective deliveries of the anticancer drug doxorubicin. This is achieved by molecular engineering, which functionalizes doxorubicin with a hydrophobic lipid tail conjugated by a solubility-promoting poly(ethylene glycol) polymer (amphiphilic doxorubicin or amph-DOX). In vivo, the amphiphile conjugated to doxorubicin exhibits a dual function: (i) it binds avidly to serum albumin and hijacks albumin’s circulating and transporting pathways, resulting in prolonged circulation in blood, increased accumulation in tumor, and reduced exposure to the heart; (ii) it also redirects doxorubicin to mitochondria by altering the drug molecule’s intracellular sorting and transportation routes. Efficient mitochondrial targeting with amph-DOX causes a significant increase of reactive oxygen species levels in tumor cells, resulting in markedly improved antitumor efficacy than the unmodified doxorubicin. Amphiphilic modification provides a simple strategy to simultaneously increase the efficacy and safety of doxorubicin in cancer chemotherapy.

Efforts for tissue engineering vascular grafts focuses on the tunica media and intima, although the tunica adventitia serves as the primary structural support for blood vessels. In surgery, during endarterectomies, surgeons can strip the vessel, leaving the adventitia as the main strength layer to close the vessel. Here, we adapted our recently developed technique of forming vascular tissue rings then stacking the rings into a tubular structure, to accommodate human fibroblasts to create adventitia vessels in 8 days. Collagen production and fibril cross-linking was augmented with TGF-β and ascorbic acid, significantly increasing tensile strength to 57.8 ± 3.07 kPa (p = 0.008). Collagen type I gel was added to the base fibrin hydrogel to further increase strength. Groups were: Fibrin only; 0.7 mg/ml COL; 1.7 mg/ml COL; and 2.2 mg/ml COL. The 0.7 mg/ml collagen rings resulted in the highest tensile strength at 77.0 ± 18.1 kPa (p = 0.015). Culture periods of 1–2 weeks resulted in an increase in extracellular matrix deposition and significantly higher failure strength but not ultimate tensile strength. Histological analysis showed the 0.7 mg/ml COL group had significantly more, mature collagen. Thus, a hydrogel of 0.7 mg/ml collagen in fibrin was ideal for creating and strengthening engineered adventitia vessels.

No abstract

Coronary artery disease remains a leading cause of death, affecting millions of Americans. With the lack of autologous vascular grafts available, engineered grafts offer great potential for patient treatment. However, engineered vascular grafts are generally not easily scalable, requiring manufacture of custom molds or polymer tubes in order to customize to different sizes, constituting a time-consuming and costly practice. Human arteries range in lumen diameter from about 2.0-38 mm and in wall thickness from about 0.5-2.5 mm. We have created a method, termed the "Ring Stacking Method," in which variable size rings of tissue of the desired cell type, demonstrated here with vascular smooth muscle cells (SMCs), can be created using guides of center posts to control lumen diameter and outer shells to dictate vessel wall thickness. These tissue rings are then stacked to create a tubular construct, mimicking the natural form of a blood vessel. The vessel length can be tailored by simply stacking the number of rings required to constitute the length needed. With our technique, tissues of tubular forms, similar to a blood vessel, can be readily manufactured in a variety of dimensions and lengths to meet the needs of the clinic and patient.

Introduction: Treatments for arteriosclerosis such as bypass surgery use vessel grafts that can cause infections in patients, with more than 50% of these grafts failing within 10 years. Discovering different methods to create vascular replacements in vitro has become one of the major focuses of tissue engineering. Material and Methods: Utilizing a novel approach termed the “Ring Stacking Method” we will create tissue resembling that of a blood vessel. With the aid of a 3D-printed PLA tubular scaffold placed in the center of a 35mm plate, we are able to create two cell ring layers. The ring layers are composed of smooth muscle cells and fibroblasts seeded onto fibrin gel, composed of 40μL of 100U/mL thrombin and 160μL of 20mg/mL fibrinogen, which have rolled towards the PLA scaffold. Results and Discussion: To evaluate our proposed technique of the Ring Stacking Method, we began by attempting to fabricate the smooth muscle layer. We used a human aortic smooth muscle cell line for relevance to cardiac bypass surgery application. The contractile properties of smooth muscle cells make them an ideal candidate for our ring formation methods. The SMC rings were subsequently stacked on top of one another using a plastic tube as an internal guide to keep the ring stack initially in place ( Fig. 1A ). The tubular SMC construct contained six ring segments and was about 10 mm in length ( Fig. 1B ). Remarkably, the lumen of the preliminary vascular construct had the ability to hold itself open, displaying structural integrity ( Fig. 1C , white arrow). Conclusion: Creation of engineered vascular replacements could eventually lead to decreased dependence on auto-graft harvesting from other parts of the body. Replacement vessels have potential to integrate into the pre-existing native tissue and promote healing and reestablishment of function of the region. The vascular construct could be applied to other ischemia disease and tissue engineering problems.

Coronary artery disease remains a leading cause of death, affecting millions of Americans. With the lack of autologous vascular grafts available, engineered grafts offer great potential for patient treatment. However, engineered vascular grafts are generally not easily scalable, requiring manufacture of custom molds or polymer tubes in order to customize to different sizes, constituting a time-consuming and costly practice. Human arteries range in lumen diameter from about 2.0-38 mm and in wall thickness from about 0.5-2.5 mm. We have created a method, termed the "Ring Stacking Method," in which variable size rings of tissue of the desired cell type, demonstrated here with vascular smooth muscle cells (SMCs), can be created using guides of center posts to control lumen diameter and outer shells to dictate vessel wall thickness. These tissue rings are then stacked to create a tubular construct, mimicking the natural form of a blood vessel. The vessel length can be tailored by simply stacking the number of rings required to constitute the length needed. With our technique, tissues of tubular forms, similar to a blood vessel, can be readily manufactured in a variety of dimensions and lengths to meet the needs of the clinic and patient.

Results: The endogenous thrombin potential (ETP) was significantly higher for Rip3 +/+ vs Rip3 À/À PPP (Fig 1). With the addition of recombinant RIP3 (rRIP3), ETP was enhanced in Rip3 À/À PPP. Furthermore, the addition of rRIP3 to Rip3 +/+ PPP was able to enhance its ETP. Using the RIP3 inhibitor GSK 0 843, the ETP of Rip3 +/+ PPP was significantly reduced compared with untreated plasma (Fig 1). There was no change in ETP when RIP3 inhibitors were used on Rip3 À/À PPP (Fig 1). Human PPP was also subjected to treatment with RIP3 inhibitor GSK 0 843. Untreated human PPP had significantly higher ETP than treated human PPP. In addition, we used our novel RIP3 inhibitor, code name C9, and found that it lowered human ETP at much lower concentrations. The in vivo effect of RIP3 on coagulation was evaluated by use of the IVC ligation models. The thrombus weight of Rip3 +/+ mice (n ¼ 6) was larger compared with Rip3 À/À mice (n ¼ 4; P < .01; Fig 2). Rip3 +/+ mice treated with C9 (n ¼ 6) vs dimethyl sulfoxide (n ¼ 5) showed a significant reduction in thrombus weight (P < .01; Fig 2).Conclusions: RIP3 appears to have a functional role in thrombus formation. The thrombin generation assays using PPP suggest that RIP3 has a functional role in coagulation outside of the cell, a novel finding. In addition, the IVC ligation models suggest that the absence and inhibition of RIP3 are capable of reducing thrombus formation in vivo. Rip3 À/À mice do not have a bleeding phenotype, suggesting that targeting RIP3 for its anticoagulative properties may be safer than current pharmaceutical treatments today.