Dormant origin firing promotes head-on transcription-replication conflicts at transcription termination sites in response to BRCA2 deficiency.

BRCA2 is a tumor suppressor protein responsible for safeguarding the cellular genome from replication stress and genotoxicity, but the specific mechanism(s) by which this is achieved to prevent early oncogenesis remains unclear. Here, we provide evidence that BRCA2 acts as a critical suppressor of head-on transcription-replication conflicts (HO-TRCs). Using Okazaki-fragment sequencing (Ok-seq) and computational analysis, we identified origins (dormant origins) that are activated near the transcription termination sites (TTS) of highly expressed, long genes in response to replication stress. Dormant origins are a source for HO-TRCs, and drug treatments that inhibit dormant origin firing led to a reduction in HO-TRCs, R-loop formation, and DNA damage. Using super-resolution microscopy, we showed that HO-TRC events track with elongating RNA polymerase II, but not with transcription initiation. Importantly, RNase H2 is recruited to sites of HO-TRCs in a BRCA2-dependent manner to help alleviate toxic R-loops associated with HO-TRCs. Collectively, our results provide a mechanistic basis for how BRCA2 shields against genomic instability by preventing HO-TRCs through both direct and indirect means occurring at predetermined genomic sites based on the pre-cancer transcriptome.

Cold Atmospheric Plasma Ameliorates Skin Diseases Involving Reactive Oxygen/Nitrogen Species-Mediated Functions.

Skin diseases are mainly divided into infectious diseases, non-infectious inflammatory diseases, cancers, and wounds. The pathogenesis might include microbial infections, autoimmune responses, aberrant cellular proliferation or differentiation, and the overproduction of inflammatory factors. The traditional therapies for skin diseases, such as oral or topical drugs, have still been unsatisfactory, partly due to systematic side effects and reappearance. Cold atmospheric plasma (CAP), as an innovative and non-invasive therapeutic approach, has demonstrated its safe and effective functions in dermatology. With its generation of reactive oxygen species and reactive nitrogen species, CAP exhibits significant efficacies in inhibiting bacterial, viral, and fungal infections, facilitating wound healing, restraining the proliferation of cancers, and ameliorating psoriatic or vitiligous lesions. This review summarizes recent advances in CAP therapies for various skin diseases and implicates future strategies for increasing effectiveness or broadening clinical indications.

In Vivo Metabolic Analysis of the Anticancer Effects of Plasma-Activated Saline in Three Tumor Animal Models.

In recent years, the emerging technology of cold atmospheric pressure plasma (CAP) has grown rapidly along with the many medical applications of cold plasma (e.g., cancer, skin disease, tissue repair, etc.). Plasma-activated liquids (e.g., culture media, water, or normal saline, previously exposed to plasma) are being studied as cancer treatments, and due to their advantages, many researchers prefer plasma-activated liquids as an alternative to CAP in the treatment of cancer. In this study, we showed that plasma-activated-saline (PAS) treatment significantly inhibited tumor growth, as compared with saline, in melanoma, and a low-pH environment had little effect on tumor growth in vivo. In addition, based on an ultra-high-performance liquid tandem chromatography-quadrupole time-of-flight mass spectrometry (UHPLC-QTOF-MS) analysis of tumor cell metabolism, the glycerophospholipid metabolic pathway was the most susceptible metabolic pathway to PAS treatment in melanoma in vitro and in vivo. Furthermore, PAS also inhibited cell proliferation in vivo in oral tongue squamous-cell cancer and non-small-cell lung cancer. There were few toxic side effects in the three animal models, and the treatment was deemed safe to use. In the future, plasma-activated liquids may serve as a potential therapeutic approach in the treatment of cancer.

Human Abuse Liability Assessment of Tobacco and Nicotine Products: Approaches for Meeting Current Regulatory Recommendations.

Many regulatory bodies now recommend that tobacco product manufacturers provide information regarding new tobacco products' abuse liability to inform regulatory authorization of currently marketed tobacco products or new product applications (including premarket tobacco product applications in the United States). In addition, the US Food and Drug Administration (FDA) recommends including this information as part of modified risk tobacco product applications. Regulators, including FDA, and many public health officials and researchers consider abuse liability assessment a model which predicts the likelihood that the use of the tobacco product would result in addiction and be used repeatedly or even sporadically resulting in undesirable effects. Abuse liability of a new, potentially reduced harm product can also inform its ability to substitute completely for more harmful tobacco products. While many methods exist, no standard tobacco product abuse liability assessment has been established. The purpose of this review is to provide background information and practical recommendations for human abuse liability testing methods to meet tobacco regulatory needs. A combination of nicotine test product pharmacokinetic, subjective effect and/or behavioral response, and physiological response data relative to comparator products with known abuse liability satisfies some regulatory requirements. Implications: This review provides a practical inspection of the current, international regulatory recommendations for abuse liability assessment of tobacco and regulatory review of such information within the United States and also recommends study designs and methods for abuse liability testing of tobacco products based on scientific and regulatory knowledge. Given that tobacco product abuse liability testing is of increasing interest to regulatory bodies globally, especially with the emergence of novel tobacco products, this timely work provides background and functional recommendations for tobacco product abuse liability testing.

Trident cold atmospheric plasma blocks three cancer survival pathways to overcome therapy resistance.

Therapy resistance is responsible for most cancer-related death and is mediated by the unique ability of cancer cells to leverage metabolic conditions, signaling molecules, redox status, and other pathways for their survival. Interestingly, many cancer survival pathways are susceptible to disturbances in cellular reactive oxygen species (ROS) and may therefore be disrupted by exogenous ROS. Here, we explore whether trident cold atmospheric plasma (Tri-CAP), a gas discharge with exceptionally low-level ROS, could inhibit multiple cancer survival pathways together in a murine cell line model of therapy-resistant chronic myeloid leukemia (CML). We show that Tri-CAP simultaneously disrupts three cancer survival pathways of redox deregulation, glycolysis, and proliferative AKT/mTOR/HIF-1α signaling in this cancer model. Significantly, Tri-CAP blockade induces a very high rate of apoptotic death in CML cell lines and in primary CD34+ hematopoietic stem and progenitor cells from CML patients, both harboring the therapy-resistant T315I mutation. In contrast, nonmalignant controls are minimally affected by Tri-CAP, suggesting it selectively targets resistant cancer cells. We further demonstrate that Tri-CAP elicits similar lethality in human melanoma, breast cancer, and CML cells with disparate, resistant mechanisms and that it both reduces tumor formation in two mouse models and improves survival of tumor-bearing mice. For use in patients, administration of Tri-CAP may be extracorporeal for hematopoietic stem cell transplantation therapy, transdermal, or through its activated solution for infusion therapy. Collectively, our results suggest that Tri-CAP represents a potent strategy for disrupting cancer survival pathways and overcoming therapy resistance in a variety of malignancies.

Plasma-activated thermosensitive biogel as an exogenous ROS carrier for post-surgical treatment of cancer.

Post-surgical residual tumor cells are the primary cause of relapse and progression of cancer but unfortunately, there are limited therapeutic options. In this work, a fillable plasma-activated biogel is produced on a thermosensitive biogel [(Poly-DL-lactide)-(poly-ethylene glycol)-(poly-DL-lactide), PLEL] with the aid of a discharge plasma for local post-operative treatment of cancer. In vivo data show that the plasma-activated PLEL biogel (PAPB) eliminates residual tumor tissues after removal surgery and also inhibits in situ recurrence while showing no evident systemic toxicity. Moreover, the PAPB possesses excellent storage capability, allows for slow release of plasma-generated reactive oxygen species (ROS), and exhibits good ROS-mediated anticancer effects in vitro. Our results reveal that the novel plasma-activated biogel is an effective therapeutic agent for local post-operative treatment of cancer.

Low-Temperature Gas Plasma Combined with Antibiotics for the Reduction of Methicillin-Resistant Staphylococcus aureus Biofilm Both In Vitro and In Vivo.

Biofilm infections in wounds seriously delay the healing process, and methicillin-resistant Staphylococcus aureus is a major cause of wound infections. In addition to inactivating micro-organisms, low-temperature gas plasma can restore the sensitivity of pathogenic microbes to antibiotics. However, the combined treatment has not been applied to infectious diseases. In this study, low-temperature gas plasma treatment promoted the effects of different antibiotics on the reduction of S. aureus biofilms in vitro. Low-temperature gas plasma combined with rifampicin also effectively reduced the S. aureus cells in biofilms in the murine wound infection model. The blood and histochemical analysis demonstrated the biosafety of the combined treatment. Our findings demonstrated that low-temperature gas plasma combined with antibiotics is a promising therapeutic strategy for wound infections.

The Antitumor Effects of Plasma-Activated Saline on Muscle-Invasive Bladder Cancer Cells In Vitro and In Vivo Demonstrate Its Feasibility as a Potential Therapeutic Approach.

Muscle-invasive bladder cancer (MIBC) is a fast-growing and aggressive malignant tumor in urinary system. Since chemotherapy and immunotherapy are only useable with a few MIBC patients, the clinical treatment of MIBC still faces challenges. Here, we examined the feasibility of plasma-activated saline (PAS) as a fledgling therapeutic strategy for MIBC treatment. Our data showed that plasma irradiation could generate a variety of reactive oxygen species (ROS) and reactive nitrogen species (RNS) in saline. In vivo tests revealed that pericarcinomatous tissue injection with PAS was effective at preventing subcutaneous bladder tumor growth, with no side effects to the visceral organs after long-term administration, as well as having no obvious influence on the various biochemistry indices of the blood in mice. The in vitro studies indicated that adding 30% PAS in cell culture media causes oxidative damage to the bladder transitional cells T24 and J82 through enhancing the intracellular ROS level, and eventually induces cancer cells' apoptosis by activating the ROS-mediated Fas/CD95 pathway. Therefore, for an intracavity tumor, these initial observations suggest that the soaking of the tumor tissue with PAS by intravesical perfusion may be a novel treatment option for bladder cancer.

Effect of He Plasma Jet Versus Surface Plasma on the Metabolites of Acute Myeloid Leukemia Cells.

Cold atmospheric plasma, including plasma jet and surface plasma, can promote the apoptosis of cancer cells without causing significant damage to surrounding normal cells, which was hopeful to be applied to the clinical cancer therapy. However, experimental plasma devices used directly to clinical experiments has challenges in technology and methods, especially the difference in killing tumor cells efficiency of these two common plasma sources. Therefore, it is great necessity to explore the differences in treating tumors between different plasma sources. This paper achieved good killing efficiency by using two kinds of cold atmospheric plasma generating devices, namely plasma jet and surface plasma treatment along acute myeloid leukemia (AML). The results showed that the He plasma jet kills leukemia cells more efficiently than surface plasma with the same voltage and frequency and the same time. By GC-TOFMS and metabolomics analysis, this paper compared the differential metabolites of leukemia cells treated by two plasma devices and the key metabolic pathways closely related to differential metabolites. Simultaneously, we found alanine, aspartate and glutamate metabolism was most correlated with a key differential metabolite, glutamine. It was found that the glutaminase activity of He plasma jet group was lower than that of surface plasma group, which might be a reason for He plasma jet group to kill tumor cells better. It was also worth noting that relative quantity of glucose metabolites of plasma jet treatment group was lower than that of surface plasma treatment group. This study provides the basis for clinical trials for future.

When upper respiratory tract infections go rogue: A case report of Arcanobacterium haemolyticum Cerebral Abscess.

Arcanobacterium haemolyticum is an extremely rare cause of cerebral abscess. We present a unique case of Arcanobacterium haemolyticum sinusitis complicated by preseptal cellulitis and cerebral abscess. The patient initially presented with pharyngitis and then developed sinus congestion, headache and facial pain. Computed tomography and magnetic resonance imaging revealed a right gyrus rectus cerebral abscess and paranasal sinus infection. The patient underwent endoscopic sinus surgery and cultures revealed Arcanobacterium haemolyticum. Repeat imaging revealed maturation and progression of intracranial abscess. The abscess was drained and patient was treated with parenteral and oral antibiotics until complete clinical and radiological remission. This case highlights the importance of recognizing Arcanobacterium haemolyticum as a cause of invasive disease in immunocompetent hosts.

Effect of cold atmospheric plasma treatment on the metabolites of human leukemia cells.

BACKGROUND: Acute myeloid leukemia (AML) is a typically fatal malignancy and new drug and treatment need to be developed for a better survival outcome. Cold atmospheric plasma (CAP) is a novel technology, which has been widely applied in biomedicine, especially in various of cancer treatment. However, the changes in cell metabolism after CAP treatment of leukemia cells have been rarely studied. METHODS: In this study, we investigated the metabolite profiling of plasma treatment on leukemia cells based on Gas Chromatography Tandem Time-of-Flight Mass Spectrometry (GC-TOFMS). Simultaneously, we conducted a series of bioinformatics analysis of metabolites and metabolic pathways with significant differences after basic data analysis. RESULTS: 800 signals were detected by GC-TOF mass-spectrometry and then evaluated using PCA and OPLS-DA. All the differential metabolites were listed and the related metabolic pathways were analyzed by KEGG pathway. The results showed that alanine, aspartate and glutamate metabolism had a significant change after plasma treatment. Meanwhile, d-glutamine and d-glutamate metabolism were significantly changed by CAP. Glutaminase activity was decreased after plasma treatment, which might lead to glutamine accumulation and leukemia cells death. CONCLUSIONS: We found the above two metabolic pathways vulnerable to plasma treatment, which might result in leukemia cells death and might be the cornerstone of further exploration of plasma treatment targets.

Efficacy of low-temperature plasma-activated gas disinfection against biofilm on contaminated GI endoscope channels.

BACKGROUND AND AIMS: It has been increasingly recognized that the safety of GI endoscopes needs to be improved by addressing the small margin of safety of high-level disinfectants (HLDs) and the failure of HLDs to clear multidrug-resistant organisms and biofilms. There is also an unmet need for effective low-temperature sterilization techniques that have a clear pathway for U.S. Food and Drug Administration clearance. Here, we report the results of our investigation of a novel argon plasma-activated gas (PAG) for disinfection and potentially sterilization of biofilm-contaminated endoscopic channels. METHODS: Test polytetrafluoroethylene channel segments were contaminated with 4-, 24- and 48-hour luminal biofilms of methicillin-resistant Staphylococcus aureus, Pseudomonas aeruginosa, or Escherichia coli and were treated by PAG flowing for up to 9 minutes. After PAG treatment, inactivation and dispersal of luminal bacterial biofilms and their regrowth in 48 hours were evaluated. Reactive species induced by PAG were measured with colorimetric probes and electron spin resonance spectrometry. Surface morphology and elemental composition of PAG-treated channel material were analyzed with scanning electron microscopy. RESULTS: PAG treatment for 9 minutes led to more than 8 log reduction of viable cells and dispersal of 24- and 48-hour luminal biofilms of all 3 bacteria and to suppression of their regrowth, whereas it resulted in little morphologic abnormalities in channel material. Ozone concentration of PAG fell to below .01 ppm within 30 seconds of switching off the plasma. PAG-treated deionized water was acidified with numerous types of reactive species, each with a concentration some 3 orders of magnitude or more below its bacterial inhibition concentration. CONCLUSIONS: PAG is capable of effectively and rapidly disinfecting luminal bacterial biofilms and offers an alternative to the step of HLDs and/or ethylene oxide in the endoscope reprocessing procedure with safety to personnel and environment.

Cold atmospheric plasma induces apoptosis of melanoma cells via Sestrin2-mediated nitric oxide synthase signaling.

Cold atmospheric plasma (CAP) represents a promising therapy for selectively cancer killing. However, the mechanism of CAP-induced cancer cell death remains unclear. Here, we identified the tumor necrosis factor-family members, especially Fas, and overloaded intracellular nitric oxide participated in CAP induced apoptosis in A375 and A875 melanoma cell lines, which was known as extrinsic apoptosis pathway. This progress was mediated by antagonistic protein of reactive oxygen species, Sestrin2. The over expression of Sestrin2 induced by plasma treatment resulted in phosphorylation of p38 mitogen-activated protein kinase (MAPK), followed by increased expression of nitric oxide synthase (iNOS), Fas and Fas ligand. Depletion of Sestrin2 reduced iNOS and Fas expression, which was associated with reduction of plasma-induced apoptosis. In contrast, inhibition of iNOS activity and phosphorylation of p38 did not alter Sestrin2 expression in plasma-treated melanoma cells. Taken together, cold atmospheric plasma increases Sestrin2 expression and further activates downstream iNOS, Fas and p38 MAPK signaling to induce apoptosis of melanoma cell lines. These findings suggest a previously unrecognized mechanism in melanoma cells response to cold atmospheric plasma therapy.

Discharge Plasma-Activated Saline Protects Against Abdominal Sepsis by Promoting Bacterial Clearance.

AIM: The aim of the present study was to investigate the effect of cold atmospheric discharge plasma-activated saline (DPAS) on abdominal sepsis. METHODS: For in vitro research, Methicillin-resistant Staphylococcus aureus (MRSA) was treated with DPAS, and the survival was detected. For in vivo research, male C57BL/6 mice were induced to sepsis by cecal ligation and puncture (CLP) and were randomly allocated into saline and DPAS control groups, CLP group, and low or high doses of DPAS (CLP + DPAS 5 and CLP + DPAS 10) groups. In experiment 1, mice were monitored for 120 h to conduct a Kaplan-Meier survival curve analysis. In experiment 2, blood, peritoneal fluid, and lung and intestinal tissues in experimental groups were collected at 4, 8, and 24 h after the CLP/sham operation to determine the severity of sepsis. RESULTS: In vitro results showed that DPAS significantly inhibited MRSA proliferation. In vivo results showed that both low and high doses of DPAS could significantly improve septic survival in the mice. DPAS treatment also significantly attenuated the lung and intestine histopathological injuries; lung wet/dry ratio; inflammatory reaction; bacterial load in the peritoneal cavity, blood, and lungs; HMGB1 and NF-κB p65 expression levels; cell apoptosis in the lung and intestine. However, there was no difference between CLP + DPAS 5 and CLP + DPAS 10 groups. CONCLUSION: In conclusion, DPAS had markedly protective effects on abdominal sepsis in mice, and the potential mechanism was associated with the ability of reactive species in DPAS to promote bacterial clearance, inhibit the inflammatory response and cell apoptosis.

Cold atmospheric plasma as a potential tool for multiple myeloma treatment.

Multiple myeloma (MM) is a fatal and incurable hematological malignancy thus new therapy need to be developed. Cold atmospheric plasma, a new technology that could generate various active species, could efficiently induce various tumor cells apoptosis. More details about the interaction of plasma and tumor cells need to be addressed before the application of gas plasma in clinical cancer treatment. In this study, we demonstrate that He+O2 plasma could efficiently induce myeloma cell apoptosis through the activation of CD95 and downstream caspase cascades. Extracellular and intracellular reactive oxygen species (ROS) accumulation is essential for CD95-mediated cell apoptosis in response to plasma treatment. Furthermore, p53 is shown to be a key transcription factor in activating CD95 and caspase cascades. More importantly, we demonstrate that CD95 expression is higher in tumor cells than in normal cells in both MM cell lines and MM clinical samples, which suggests that CD95 could be a favorable target for plasma treatment as it could selectively inactivate myeloma tumor cells. Our results illustrate the molecular details of plasma induced myeloma cell apoptosis and it shows that gas plasma could be a potential tool for myeloma therapy in the future.

Cold atmospheric-pressure plasma induces DNA-protein crosslinks through protein oxidation.

Reactive oxygen and nitrogen species (ROS and RNS) generated by cold atmospheric-pressure plasma could damage genomic DNA, although the precise types of these DNA damage induced by plasma are poorly characterized. Understanding plasma-induced DNA damage will help to elucidate the biological effect of plasma and guide the application of plasma in ROS-based therapy. In this study, it was shown that ROS and RNS generated by physical plasma could efficiently induce DNA-protein crosslinks (DPCs) in bacteria, yeast, and human cells. An in vitro assay showed that plasma treatment resulted in the formation of covalent DPCs by activating proteins to crosslink with DNA. Mass spectrometry and hydroperoxide analysis detected oxidation products induced by plasma. DPC formation were alleviated by singlet oxygen scavenger, demonstrating the importance of singlet oxygen in this process. These results suggested the roles of DPC formation in DNA damage induced by plasma, which could improve the understanding of the biological effect of plasma and help to develop a new strategy in plasma-based therapy including infection and cancer therapy.

Antimicrobial Efficacy and Safety of a Novel Gas Plasma-Activated Catheter Lock Solution.

Antimicrobial lock solutions are important for prevention of microbial colonization and infection of long-term central venous catheters. We investigated the efficacy and safety of a novel antibiotic-free lock solution formed from gas plasma-activated disinfectant (PAD). Using a luminal biofilm model, viable cells of methicillin-resistant Staphylococcus aureus, Staphylococcus epidermidis, Pseudomonas aeruginosa, and Candida albicans in mature biofilms were reduced by 6 to 8 orders of magnitude with a PAD lock for 60 min. Subsequent 24-h incubation of PAD-treated samples resulted in no detectable regrowth of viable bacteria or fungi. As a comparison, the use of a minocycline-EDTA-ethanol lock solution for 60 min led to regrowth of bacteria and fungi, up to 107 to 109 CFU/ml, in 24 h. The PAD lock solution had minimal impact on human umbilical vein endothelial cell viability, whereas the minocycline-EDTA-ethanol solution elicited cell death in nearly half of human endothelial cells. Additionally, PAD treatment caused little topological change to catheter materials. In conclusion, PAD represents a novel antibiotic-free, noncytotoxic lock solution that elicits rapid and broad-spectrum eradication of biofilm-laden microbes and shows promise for the prevention and treatment of intravascular catheter infections.

Gas Plasma Pre-treatment Increases Antibiotic Sensitivity and Persister Eradication in Methicillin-Resistant Staphylococcus aureus.

Methicillin-resistant Staphylococcus aureus (MRSA) is a major cause of serious nosocomial infections, and recurrent MRSA infections primarily result from the survival of persister cells after antibiotic treatment. Gas plasma, a novel source of ROS (reactive oxygen species) and RNS (reactive nitrogen species) generation, not only inactivates pathogenic microbes but also restore the sensitivity of MRSA to antibiotics. This study further found that sublethal treatment of MRSA with both plasma and plasma-activated saline increased the antibiotic sensitivity and promoted the eradication of persister cells by tetracycline, gentamycin, clindamycin, chloramphenicol, ciprofloxacin, rifampicin, and vancomycin. The short-lived ROS and RNS generated by plasma played a primary role in the process and induced the increase of many species of ROS and RNS in MRSA cells. Thus, our data indicated that the plasma treatment could promote the effects of many different classes of antibiotics and act as an antibiotic sensitizer for the treatment of antibiotic-resistant bacteria involved in infectious diseases.

Alteration of metabolite profiling by cold atmospheric plasma treatment in human myeloma cells.

BACKGROUND: Despite new progress of chemotherapy in multiple myeloma (MM) clinical treatment, MM is still a refractory disease and new technology is needed to improve the outcomes and prolong the survival. Cold atmospheric plasma is a rapidly developed technology in recent years, which has been widely applied in biomedicine. Although plasma could efficiently inactivate various tumor cells, the effects of plasma on tumor cell metabolism have not been studied yet. METHODS: In this study, we investigated the metabolite profiling of He plasma treatment on myeloma tumor cells by gas-chromatography time-of-flight (GC-TOF) mass-spectrometry. Meanwhile, by bioinformatic analysis such as GO and KEGG analysis we try to figure out the metabolism pathway that was significantly affected by gas plasma treatment. RESULTS: By GC-TOF mass-spectrometry, 573 signals were detected and evaluated using PCA and OPLS-DA. By KEGG analysis we listed all the differential metabolites and further classified into different metabolic pathways. The results showed that beta-alanine metabolism pathway was the most significant change after He gas plasma treatment in myeloma cells. Besides, propanoate metabolism and linoleic acid metabolism should also be concerned during gas plasma treatment of cancer cells. CONCLUSIONS: Cold atmospheric plasma treatment could significantly alter the metabolite profiling of myeloma tumor cells, among which, the beta-alanine metabolism pathway is the most susceptible to He gas plasma treatment.

Intracellular ROS mediates gas plasma-facilitated cellular transfection in 2D and 3D cultures.

This study reports the potential of cold atmospheric plasma (CAP) as a versatile tool for delivering oligonucleotides into mammalian cells. Compared to lipofection and electroporation methods, plasma transfection showed a better uptake efficiency and less cell death in the transfection of oligonucleotides. We demonstrated that the level of extracellular aqueous reactive oxygen species (ROS) produced by gas plasma is correlated with the uptake efficiency and that this is achieved through an increase of intracellular ROS levels and the resulting increase in cell membrane permeability. This finding was supported by the use of ROS scavengers, which reduced CAP-based uptake efficiency. In addition, we found that cold atmospheric plasma could transfer oligonucleotides such as siRNA and miRNA into cells even in 3D cultures, thus suggesting the potential for unique applications of CAP beyond those provided by standard transfection techniques. Together, our results suggest that cold plasma might provide an efficient technique for the delivery of siRNA and miRNA in 2D and 3D culture models.