Direct Needle Injection Research Articles

Regional infusion of a class C TLR9 agonist enhances liver tumor microenvironment reprogramming and MDSC reduction to improve responsiveness to systemic checkpoint inhibition

Myeloid-derived suppressor cells (MDSCs) expand in response to malignancy and suppress responsiveness to immunotherapy, including checkpoint inhibitors (CPIs). Within the liver, MDSCs have unique immunosuppressive features. While TLR9 agonists have shown promising activities in enhancing CPI responsiveness in superficial tumors amenable to direct needle injection, clinical success for liver tumors with TLR9 agonists has been limited by delivery challenges. Here, we report that regional intravascular infusion of ODN2395 into mice with liver metastasis (LM) partially eliminated liver MDSCs and reprogrammed residual MDSC. TLR9 agonist regional infusion also induced an increase in the M1/M2 macrophage ratio. Enhanced TLR9 signaling was demonstrated by an increased activation of in NFκB (pP65) and production of IL6 compared with systemic infusion. Further, PBMC-derived human MDSCs express TLR9, and treatment with class C TLR9 agonists (ODN2395 and SD101) reduced the expansion of MDSC population. TLR9 stimulation induced MDSC apoptosis and increased the M1/M2 macrophage ratio. Regional TLR9 agonist infusion along with systemic anti-PD-1 therapy improved control of LM. With effective delivery, TLR9 agonists have the potential to favorably reprogram the liver TME through reduction of MDSCs and favorable macrophage polarization, which may improve responsiveness to systemic CPI therapy.

Abstract No. 333 Improved delivery of a TLR-9 agonist to liver tissue by intravascular pressure enabled drug delivery (PEDD) compared with direct needle injection

Direct injection of the TLR-9 agonist SD-101 oligonucleotide has shown promising results for the treatment of superficial cutaneous melanoma metastatic lesions in combination with checkpoint inhibition. However, this mode of delivery is limited to easily discernable tumors located in superficial locations. Here we compare local infusion of labeled SD-101 into hepatic tissue using the Pressure Enabled Drug Delivery (PEDD) method vs. direct needle injection. PEDD has been shown to increase the concentration and penetration of therapeutics into tumors, while limiting exposure to off-target tissue.

Abstract 1793: Regional administration of class C CpG Oligodeoxynucleotides results in superior intrahepatic TLR9 activation and immunomodulation compared to systemic infusion

Abstract CpG oligodeoxynucleotides (ODNs) are TLR9 agonists (TLR9As) that stimulate pDC and B cells. Yet, multiple other cell types in the liver express TLR9. The effect of TLR9A in reshaping the tumor microenvironment (TME), particularly in the myeloid-derived suppressor cells (MDSC) compartment, is yet to be determined. Clinical success of TLR9As has been limited by toxicity when given systemically and delivery limitations when administered by direct needle injection. Here, we hypothesized that regional intra-vascular infusion of TRL9As would reprogram the liver MDSC compartment to enable immune control of liver metastases (LM). In this study, we evaluated the effect of class C ODN-2395 as TLR9A in inhibiting LM progression and its impact on the liver MDSC population. C57/BL6 mice were challenged with 2.5e6 MC38-CEA-Luc cells via intra-splenic route. After a week, mice were treated with 1, 3, 10 or 30 µg ODN2395 via PV or IV (30 µg). Bioluminescence was measured at 24 and 48 hrs post-ODN administration. CD45+ cells were isolated, and FACS analysis was performed to quantify MDSCs, monocytic MDSCs (M-MDSC), and M1-macrophage subsets, along with downstream signaling. We found that 30 µg ODN2395 delivered via PV compared to IV reduced tumor burden at 24 hrs and persisted up to 48 hrs. Also, 30 µg ODN2395 infused via PV was optimal in controlling MDSC, M-MDSC cells and polarized CD11b+F4/80+ monocytic cells towards pro-inflammatory/anti-tumorigenic M1 macrophage. Using an NFκB-dependent soluble alkaline phosphatase assay (SEAP), we determined that ODN2395 dose-dependently enhanced NFκB transcription factor activity (p<0.001). Western Blot data of tumor lysates show that ODN delivery by PV increased NFκB (pP65) activity and production of IL-6, significantly relative to IV. Overall, our data suggest that the regional delivery of a TLR9A has the potential to provide superior modulation of the TME within the liver. Table:Readout30µg IV30µg PVP valuenBioluminescence-24 h (photon/s)4e71e7<0.0113Bioluminescence-48h (photon/s)7e72e7<0.0513FACSCD11b+Gr1+ cells/CD45+ NPC (MDSC)29.70 %20.75%<0.0113CD11b+LY6C+ (M-MDSC)60.03%38.98%<0.0113CD11b+F4/80+CD38+EGR2-(M1 macrophage)34.82%58.20%<0.0113Western BlottingpNFκB/NFκB (pP65s276/pP65): Fold change0.862.028<0.016IL6/GAPDH Fold change12.387<0.056 Citation Format: Chandra Ghosh, Kara Heatherton, Kyle O'Connell, Jason Laporte, Prajna Guha, David Jaroch, Bryan Cox, Steven C. Katz. Regional administration of class C CpG Oligodeoxynucleotides results in superior intrahepatic TLR9 activation and immunomodulation compared to systemic infusion [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1793.

In silico study of enhanced permeation and retention effect and hyperthermia of porous tumor

Nanotechnology has recently gained fame for its extensive use in biomedical applications particularly in magnetic fluid hyperthermia (MFH) of tumors. The magnetic nanoparticles (MNPs) are usually injected into the tumor either intravenously or through direct needle injection. Depending on the location of the tumor, the needle approach may not be appropriate and in the case, when the nanoflow rate is higher, it may produce cracks in the tumor. In this scenario, the intravenous approach following the enhanced permeation and retention effect (EPR) effect proves advantageous. In this paper, we have simulated the EPR effect of nanofluid flowing from blood vessels to the tumor through epithelial cells spacing and then its diffusion in the tumor interstitium using COMSOL Multiphysics. The velocity in the blood vessel and diffusion in the tumor have been simulated and analyzed using Finite Element Method (FEM) based models of Navier-Stokes equations and convection-diffusion equation. The simulation results show that the velocity and concentration are higher in the blood vessel and it decreases slowly while moving through epithelial spacing to the tumor interstitium. The heat transfer in the tumor interstitium is simulated and analyzed for temperature distribution quantitatively.

Therapeutic Efficacy of onabotulinumtoxinA Delivered Using Various Approaches in Sensory Bladder Disorder

Cystoscopic onabotulinumtoxinA (onaBoNTA) intradetrusor injection is an efficient and durable modality for treating sensory bladder disorders. However, the inconvenience of using the cystoscopic technique and anesthesia, and the adverse effects of direct needle injection (e.g., haematuria, pain, and infections) have motivated researchers and clinicians to develop diverse injection-free procedures to improve accessibility and prevent adverse effects. However, determining suitable approaches to transfer onaBoNTA, a large molecular and hydrophilic protein, through the impermeable urothelium to reach therapeutic efficacy remains an unmet medical need. Researchers have provided potential solutions in three categories: To disrupt the barrier of the urothelium (e.g., protamine sulfate), to increase the permeability of the urothelium (e.g., electromotive drug delivery and low-energy shock wave), and to create a carrier for transportation (e.g., liposomes, thermosensitive hydrogel, and hyaluronan-phosphatidylethanolamine). Thus far, most of these novel administration techniques have not been well established in their long-term efficacy; therefore, additional clinical trials are warranted to validate the therapeutic efficacy and durability of these techniques. Finally, researchers may make progress with new combinations or biomaterials to change clinical practices in the future.

Langendorff-Free Isolation and Propagation of Adult Mouse Cardiomyocytes.

Isolation of healthy, intact cardiomyocytes from the adult mouse heart for cardiac research is challenging. Traditional protocols depend upon specialized Langendorff apparatus, which requires extensive training and presents significant technical and logistical barriers. Described here is a much simplified technique, introducing optimized dissociation buffers to the heart by direct needle injection into the left ventricle, allowing deep myocardial perfusion and the isolation of high yields of viable, rod-shaped cardiomyocytes, using only standard surgical and laboratory equipment. This method also permits the concurrent isolation of cardiac non-myocyte cellular populations.

Gadolinium Augmentation of Myocardial Tissue Heating During Radiofrequency Ablation

ObjectivesThis study hypothesized that a metal already commonly used in medical procedures, gadolinium (Gd), will augment radiofrequency (RF) thermal injury and affect cardiac ablation lesions. BackgroundEnhancement of RF ablation using metallic particles has been proposed for ablation of tumors. MethodsA series of ablation lesions were delivered at variable power using an ex vivo model. Tissue temperatures and lesion characteristics were analyzed. Ablation in a porcine in vivo model after direct needle injection of the myocardium with Gd or after systemic administration of Gd encased in heat sensitive liposomes was also performed and compared to control values. ResultsAblation after Gd infiltration of myocardial tissue resulted in significantly larger lesions at both low- and high-power settings. Larger impedance changes were observed during ablation of Gd-treated myocardium. In vivo ablation using a force-sensing irrigated tip catheter resulted in enhanced lesion sizes after Gd injection without a higher incidence of steam pops or perforation. Systemic administration of liposomal Gd with local release by RF heating did not result in larger ablation sizes. ConclusionsGd can be used to enhance RF ablation lesions. In both ex vivo studies with a 4-mm ablation catheter under power control and in vivo findings with an irrigated tip catheter, ablation of myocardium infiltrated with Gd resulted in larger lesions, with altered RF electrical and thermal characteristics. More research is needed to refine the potential for Gd facilitation of RF ablation. The use of systemic heat-sensitive liposomes containing Gd with targeted release by RF heating did not affect lesion size.

Local Chemotherapy for Pancreatic Cancer: Potential of Injectable Drug-Loaded Microparticles

Introduction: A key limitation of systemic delivery of chemotherapy in pancreatic adenocarcinoma (PDAC) is poor drug penetrance to the tumor due to the surrounding desmoplastic reaction. Systemic side effects also interfere with treatment continuity. These can be circumvented with a local delivery method via endoscopic ultrasound (EUS) fine needle injection. Poly(lactic-co-glycolic acid) (PLGA) is an FDA-approved polymer that can be utilized to create spherical microparticles (MPs) containing encapsulated chemotherapy that is slowly eluted with particle degradation. Methods: Local delivery of gemcitabine encapsulated within MPs (GMPs) to the the pancreas was previously explored by our lab in a 3-phase experiment. In phase I, blank MPs were injected into mouse pancreata to assess for adverse effects from the polymer and none were noted. In phase II, GMPs were injected into pancreatic tumors grown in nude mice. Injected tumors showed evidence of apoptosis compared to control pancreatic tumors. In Phase III, EUS-guided fine needle injection of GMPs into the porcine pancreas was performed. Survival was assessed for 14 days. GMPs were not localized in pancreatic tissue, calling into question if GMPs entered pancreatic tissue upon injection. This study evaluates if MPs containing fluorescently labelled protein can be localized in porcine pancreatic tissue after direct needle injection. We also present a methodology of simultaneous fluorescent labeling of PLGA and encapsulation of gemcitabine. Pancreata were harvested from pigs within 1 hour of euthanasia and placed in PBS for transport. MPs were injected into the pancreas aiming to approximate EUS conditions. Results: The tissue was H&E stained for light microscope visualization and MPs were visualized in the parenchyma and within interlobular septae. MPs were not visualized in control sections of porcine pancreatic tissue. Albumin-fluorescein isothiocyanate (BSA-FITC) was encapsulated in MPs and the injection experiments were repeated to confirm localization. Conclusion: This provided preliminary evidence that MPs enter the pancreatic tissue after injection and can be visualized. Thereafter, BSA-FITC was covalently bonded to the PLGA polymer allowing concurrent fluorescence and gemcitabine encapsulation. Visualization of the MPs in the porcine pancreas is rationale for repeating EUS fine needle injection into the pig pancreas, with the goal of developing a versatile and practical methodology for localized chemotherapy delivery to human pancreatic tumors.

Identifying unrecognized collecting system entry and the integrity of repair during open partial nephrectomy: comparison of two techniques

To compare retrograde dye injection through an externalized ureteral catheter with direct needle injection of dye into proximal ureter for identification of unrecognized collecting system disruption and integrity of subsequent repair during open partial nephrectomy. We retrospectively reviewed the records of 259 consecutive patients who underwent open partial nephrectomy. Externalized ureteral catheters were placed preoperatively in 110 patients (Group 1); needle injection of methylene blue directly into proximal ureter was used in 120 patients (Group 2). No assessment of the collecting system was performed in 29 patients (Group 3). We compared intraoperative parameters, tumor characteristics, collecting system entry and incidence of urine leaks among the three groups. The mean tumor diameter was 3.1 cm in Group 1, 3.6cm in Group 2, and 3.8 cm in Group 3 (p = 0.04); mean EBL 320cc, 351 cc and 376cc (p = 0.5); mean operative time 193.5 minutes, 221 minutes and 290 minutes (p < 0.001). Collecting system entry was recognized in 63%, 76% and 38% of cases in Groups 1, 2 and 3 respectively. (p = 0.07). Postoperative urine leaks requiring some form of management occurred in 11 patients from group 1 and 6 from group 2. (p = 0.2). No patient in Group 3 developed a urinary leak. Identification of unrecognized collecting system disruption as well as postoperative urine leak rate in patients undergoing partial nephrectomy were not influenced by the intraoperative technique of identifying unrecognized collecting system entry. Postoperative urine leaks are uncommon despite recognized collecting system disruption in the majority of patients.

Ultrasound‐guided injection of the median artery in the standing sedated horse

Injection of the median artery of horses leads to better distribution and persistence of mesenchymal stem cells than i.v. regional limb perfusion. Due to technical difficulties, intra-arterial injections thus far have only been performed under general anaesthesia. To assess the feasibility of injection of the median artery in standing sedated horses. Experimental study. Six horses were included in the study. After median and ulnar regional analgesia, radiographic contrast material was injected in the median artery of both front limbs, using a catheter in one limb and a direct needle injection in the other. Ultrasound guidance was used for catheter and needle placement. Radiographs were obtained for confirmation of successful injection. Post procedural ultrasound examination was performed to assess vascular compromise. Catheter placement was successful in all 6 limbs, but in one limb injection was not possible due to arterial spasm. Movement of the limbs after the initial injection resulted in loss of functionality of the catheter in 2 other horses. Direct needle injection was successful on all 6 limbs, with periarterial extravasation observed in 2 limbs. No clinical complications were observed. Injection of the median artery can be performed in standing horses under sedation. Direct needle injection is a more practical technique than catheterisation, as it is easier to perform and less likely to induce arterial spasm. Periarterial extravasation remains a possible limitation of the technique. Intra-arterial injections may be useful for administration of therapeutic agents such as mesenchymal stem cells on standing sedated horses.

Evaluation of Thiol-Modified Hyaluronan and Elastin-Like Polypeptide Composite Augmentation in Early-Stage Disc Degeneration

An in vitro biomechanical and imaging study generated from an in vivo porcine model of early stage degenerative disc disease was used to evaluate mechanical property restoration, comparing 2 minimally invasive injection techniques. To evaluate the ability of an injectable hydrogel to restore the mechanical properties of spinal motion segments with early stage disc degeneration, comparing 2 minimally invasive injection techniques. Treatment of early-stage disc degeneration may benefit from a combination of tissue engineering and minimally invasive therapeutic approaches. A recently developed hydrogel, thiol-modified hyaluronan elastin-like polypeptide (TMHA/EP) composite, has demonstrated potential as an injectable nucleus replacement. From a total of thirteen 35-kg Yorkshire boars, early-stage lumbar disc degeneration was introduced into 10 pigs via injection of chondroitinase ABC. After degeneration, 8 pigs received TMHA/EP augmentation; 1 disc via direct needle injection and a second using a modified kyphoplasty approach. High-resolution magnetic resonance images were acquired of the excised spinal motion segments, followed by biomechanical testing in axial compression, flexion-extension, lateral bending, and torsion. The degenerate control motion segments were generally less stiff and more flexible than healthy controls. The injection of TMHA/EP into the degenerated nucleus produced similar mechanical stiffness to healthy controls. The direct-injected discs showed a dispersive pattern of TMHA/EP within the nucleus, whereas the modified kyphoplasty method yielded a bolus of hydrogel. Yet, mechanical behavior was comparable considering the 2 minimally invasive augmentation techniques. The TMHA/EP composite can restore initial mechanical behavior in early-stage disc degeneration. Although both augmentation methods yielded mechanical properties comparable with healthy controls, direct injection represents a simpler technique, uses a smaller-gauge needle, does not introduce air into the disc, and yields a dispersive pattern that may be beneficial for future delivery of cells or growth factors.

Peritoneal Changes after Exposure to Sterile Solutions by Catheter

Most current animal models that are used to study effects of long-term peritoneal exposure to dialysis solutions use an indwelling catheter for daily injections. It was hypothesized that the presence of a foreign body in the peritoneal cavity (PC) might alter the inflammatory response to the solutions and that the response would depend on exposure duration. For addressing these, long-term injections were carried out for 2 to 8 wk in 90 Sprague-Dawley rats: 40 via a subcutaneous port connected to a silicone catheter tunneled to the PC, 40 via direct needle injection, and 10 noninjected, age-control rats. Daily volumes were 30 to 40 ml of filter-sterilized, bicarbonate-buffered solutions that contained 4% dextrose. After 2, 4, 6, and 8 wk, anesthetized rats underwent transport experiments with a chamber affixed to the abdominal wall to determine mass transfer coefficients of mannitol (MTC(mannitol)) and albumin (MTC(BSA)), osmotic filtration flux (J(osm)), and hydrostatic pressure-driven flux. After the rats were killed, tissues were collected for measurement of peritoneal thickness, vascular density, and immunohistochemical staining. ANOVA demonstrated significant (P < 0.01) differences in thickness, vessel density, MTC(mannitol), and MTC(BSA) among the groups at the various time intervals and in overall means. Differences among the groups were less pronounced for hydrostatic pressure-driven flux and J(osm). Vessel density, MTC(mannitol), MTC(BSA), and J(osm) were dependent on injection duration (P < 0.01). There were marked differences between the needle injection and catheter injection groups at various intervals in the expression of three cytokines. It is concluded that the histologic and functional response depends on the duration of injection with animals that are exposed for as little as 2 wk demonstrating alterations. These findings confirm the hypothesis that the presence of a PC catheter increases inflammatory response to sterile solutions as evidenced by the structural and functional changes in the peritoneal barrier.

Detection of diaphragmatic disruptions by peritoneoscintigraphy using technetium-99M diethylene-triamine pentacetic acid.

Intraperitoneal injection of a selected radiopharmaceutical results in the diffusion of radioactive material throughout the peritoneum. A diaphragmatic injury should theoretically result in the diffusion of the radioactive material into the chest. To test this hypothesis, Technetium-99m diethylene-triamine pentacetic acid (Tc-99m DTPA) was administered intraperitoneally by either direct needle injection or catheter into 18 rabbits. Four of the rabbits served as controls and did not have any diaphragmatic injury. Fourteen rabbits had surgically induced diaphragmatic tears of varying size (1/4 to 1 cm) after thoracotomy. Four of the 14 rabbits were dropped from the study because they had inadequate peritoneal injections of the radiopharmaceutical. The remaining ten rabbits showed peritoneoscintigraphic evidence of diaphragmatic injury either by showing passage of the radiotracer into the chest, demonstrating the site of injury as a focal region of increased radiotracer uptake, or showing both of these features. Peritoneoscintigraphy appears to be a potentially useful modality in the detection of diaphragmatic injury.

Model of experimental chronic osteomyelitis in rats.

We describe here a Sprague-Dawley rat model for chronic osteomyelitis. Staphylococcus aureus and sodium morrhuate were implanted by either microdrilling or direct needle injection into the tibiae of rats. Of 107 rats, 87 (81%) developed osteomyelitis when a high-speed drill was used for implantation, and 27 (51%) of 53 rats developed osteomyelitis by direct needle inoculation (chi square = 9.81, P less than 0.01). Demonstrated histopathological changes included the presence of resorption bays filled with osteoclasts. Quantitative microbiological monitoring of tibial count confirmed disease chronicity, yielding stable numbers of CFU (10(6.29 +/- 0.27) ) of S. aureus over 70 days. Infected animals became anemic and lost weight. The erythrocyte sedimentation rates and leukocyte counts were not elevated. Roentgenograms provided the best correlation with the number of organisms in infected tibiae (r2 = 0.80). Rats with infected tibiae were treated with either oxacillin (120 mg/kg per day) or ceftriaxone (50 mg/kg per day). Treatment over 14 or 28 days reduced S. aureus counts in tibiae but did not reliably sterilize infected bones, suggesting that this model was resistant to prolonged antimicrobial therapy.