Phytocompounds and Nanoformulations for Anticancer Therapy: A Review.

Cancer is a complex disease that affects millions of people and remains a major public health problem worldwide. Conventional cancer treatments, including surgery, chemotherapy, immunotherapy, and radiotherapy, have limited achievements and multiple drawbacks, among which are healthy tissue damage and multidrug-resistant phenotype onset. Increasing evidence shows that many plants' natural products, as well as their bioactive compounds, have promising anticancer activity and exhibit minimal toxicity compared to conventional anticancer drugs. However, their widespread use in cancer therapy is severely restricted by limitations in terms of their water solubility, absorption, lack of stability, bioavailability, and selective targeting. The use of nanoformulations for plants' natural product transportation and delivery could be helpful in overcoming these limitations, thus enhancing their therapeutic efficacy and providing the basis for improved anticancer treatment strategies. The present review is aimed at providing an update on some phytocompounds (curcumin, resveratrol, quercetin, and cannabinoids, among others) and their main nanoformulations showing antitumor activities, both in vitro and in vivo, against such different human cancer types as breast and colorectal cancer, lymphomas, malignant melanoma, glioblastoma multiforme, and osteosarcoma. The intracellular pathways underlying phytocompound anticancer activity and the main advantages of nanoformulation employment are also examined. Finally, this review critically analyzes the research gaps and limitations causing the limited success of phytocompounds' and nanoformulations' clinical translation.

A natural product, voacamine, sensitizes paclitaxel-resistant human ovarian cancer cells.

Most women with ovarian cancer are treated with chemotherapy before or after surgery. Unfortunately, chemotherapy treatment can cause negative side effects and the onset of multidrug resistance (MDR). The aim of this study is to evaluate the chemosensitizing effect of a natural compound, voacamine (VOA), in ovarian (A2780 DX) and colon (LoVo DX) cancer drug-resistant cell lines which overexpress P-glycoprotein (P-gp), in combination with paclitaxel (PTX), or doxorubicin (DOX) or 5-fluorouracil (5-FU). VOA, a bisindole alkaloid extracted from Peschiera fuchsiaefolia, has already been shown to be effective in enhancing the effect of doxorubicin, because it interferes with the P-gp function. Ovarian cancer cytotoxicity test shows that single treatments with VOA, DOX and PTX do not modify cell viability, while pretreatment with VOA, and then PTX or DOX for 72 h, induces a decrease. In colon cancer, since 5-FU is not a-substrate for P-gp, VOA has no sensitizing effect while in VOA + DOX there is a decrease in viability. Annexin V/PI test, cell cycle analysis, activation of cleaved PARP1 confirm that VOA plus PTX induce apoptotic cell death. Confocal microscopy observations show the different localization of NF-kB after treatment with VOA + PTX, confirming the inhibition of nuclear translocation induced by VOA pretreatment. Our data show the specific effect of VOA which only works on drugs known to be substrates of P-gp.

Prunus spinosa Extract Sensitized HCT116 Spheroids to 5-Fluorouracil Toxicity, Inhibiting Autophagy.

Autophagy is a lysosomal degradation and recycling process involved in tumor progression and drug resistance. The aim of this work was to inhibit autophagy and increase apoptosis in a 3D model of human colorectal cancer by combined treatment with our patented natural product Prunus spinosa + nutraceutical activator complex (PsT + NAC®) and 5-fluorouracil (5-FU). By means of cytotoxic evaluation (MTT assay), cytofluorimetric analysis, light and fluorescence microscopy investigation and Western blotting evaluation of the molecular pathway PI3/AKT/mTOR, Caspase-9, Caspase-3, Beclin1, p62 and LC3, we demonstrated that the combination PsT + NAC® and 5-FU significantly reduces autophagy by increasing the apoptotic phenomenon. These results demonstrate the importance of using non-toxic natural compounds to improve the therapeutic efficacy and reduce the side effects induced by conventional drugs in human colon cancer.

Label-free cell based impedance measurements of ZnO nanoparticles-human lung cell interaction: a comparison with MTT, NR, Trypan blue and cloning efficiency assays.

BACKGROUND: There is a huge body of literature data on ZnOnanoparticles (ZnO NPs) toxicity. However, the reported results are seen to be increasingly discrepant, and deep comprehension of the ZnO NPs behaviour in relation to the different experimental conditions is still lacking. A recent literature overview emphasizes the screening of the ZnO NPs toxicity with more than one assay, checking the experimental reproducibility also versus time, which is a key factor for the robustness of the results. In this paper we compared high-throughput real-time measurements through Electric Cell-substrate Impedance-Sensing (ECIS®) with endpoint measurements of multiple independent assays. RESULTS: ECIS-measurements were compared with traditional cytotoxicity tests such as MTT, Neutral red, Trypan blue, and cloning efficiency assays. ECIS could follow the cell behavior continuously and noninvasively for days, so that certain long-term characteristics of cell proliferation under treatment with ZnO NPs were accessible. This was particularly important in the case of pro-mitogenic activity exerted by low-dose ZnO NPs, an effect not revealed by endpoint independent assays. This result opens new worrisome questions about the potential mitogenic activity exerted by ZnO NPs, or more generally by NPs, on transformed cells. Of importance, impedance curve trends (morphology) allowed to discriminate between different cell death mechanisms (apoptosis vs autophagy) in the absence of specific reagents, as confirmed by cell structural and functional studies by high-resolution microscopy. This could be advantageous in terms of costs and time spent. ZnO NPs-exposed A549 cells showed an unusual pattern of actin and tubulin distribution which might trigger mitotic aberrations leading to genomic instability. CONCLUSIONS: ZnO NPs toxicity can be determined not only by the intrinsic NPs characteristics, but also by the external conditions like the experimental setting, and this could account for discrepant data from different assays. ECIS has the potential to recapitulate the needs required in the evaluation of nanomaterials by contributing to the reliability of cytotoxicity tests. Moreover, it can overcome some false results and discrepancies in the results obtained by endpoint measurements. Finally, we strongly recommend the comparison of cytotoxicity tests (ECIS, MTT, Trypan Blue, Cloning efficiency) with the ultrastructural cell pathology studies.

A harmonized and standardized in vitro approach produces reliable results on silver nanoparticles toxicity in different cell lines.

Despite the widespread use of silver nanoparticles (AgNPs) in different fields and the amount of investigations available, to date, there are many contradictory results on their potential toxicity. In the present study, extensively characterized 20-nm AgNPs were investigated using optimized protocols and standardized methods to test several toxicological endpoints in different cell lines. The agglomeration/aggregation state of AgNPs in culture media was measured by dynamic light scattering (DLS). DNA and chromosomal damage on BEAS-2B and RAW 264.7 cells were evaluated by comet and micronucleus assays, while oxidative DNA damage by modified comet assay and 8-oxodG/8-oxodA detection. We also investigated immunotoxicity and immunomodulation by cytokine release and NO production in RAW 264.7 and MH-S cells, with or without lipopolysaccharide (LPS) stimulus. Transmission electron microscope (TEM) analysis was used to analyze cellular uptake of AgNPs. Our results indicate different values of AgNPs hydrodynamic diameter depending on the medium, some genotoxic effect just on BEAS-2B and no or slight effects on function of RAW 264.7 and MH-S in absence or presence of LPS stimulus. This study highlights the relevance of using optimized protocols and multiple endpoints to analyze the potential toxicity of AgNPs and to obtain reliable and comparable results.

Targeting Autophagy to Overcome Human Diseases.

Autophagy is an evolutionarily conserved cellular process, through which damaged organelles and superfluous proteins are degraded, for maintaining the correct cellular balance during stress insult. It involves formation of double-membrane vesicles, named autophagosomes, that capture cytosolic cargo and deliver it to lysosomes, where the breakdown products are recycled back to cytoplasm. On the basis of degraded cell components, some selective types of autophagy can be identified (mitophagy, ribophagy, reticulophagy, lysophagy, pexophagy, lipophagy, and glycophagy). Dysregulation of autophagy can induce various disease manifestations, such as inflammation, aging, metabolic diseases, neurodegenerative disorders and cancer. The understanding of the molecular mechanism that regulates the different phases of the autophagic process and the role in the development of diseases are only in an early stage. There are still questions that must be answered concerning the functions of the autophagy-related proteins. In this review, we describe the principal cellular and molecular autophagic functions, selective types of autophagy and the main in vitro methods to detect the role of autophagy in the cellular physiology. We also summarize the importance of the autophagic behavior in some diseases to provide a novel insight for target therapies.

Cytotoxic and Apoptotic Activities of Prunus spinosa Trigno Ecotype Extract on Human Cancer Cells.

The aim of this work was to demonstrate that a natural compound, not-toxic to normal cells, has cytotoxic and sensitizing effects on carcinoma cells, with the final goal of combining it with chemotherapeutic drugs to reduce the overall dose. Prunus spinosa Trigno ecotype (PsT) drupe extract with a nutraceutical activator complex (NAC) made of amino acids, vitamins and mineral salt blends, has shown in vitro anticancer activity. The cytotoxic effect of (PsT + NAC)® has been evaluated on human cancer cells, with an initial screening with colorectal, uterine cervical, and bronchoalveolar cells, and a subsequent focus on colon carcinoma cells HCT116 and SW480. The viability reduction of HCT116 and SW480 after treatment with (PsT 10 mg/mL + NAC)® was about 40% (p < 0.05), compared to control cells. The cell's survival reduction was ineffective when the drug vehicle (NAC) was replaced with a phosphate buffer saline (PBS) or physiological solution (PS). The flow cytometry evaluation of cancer cells' mitochondrial membrane potential showed an increase of 20% depolarized mitochondria. Cell cycle analysis showed a sub G1 (Gap 1 phase) peak appearance (HCT116: 35.1%; SW480: 11.6%), indicating apoptotic cell death induction that was confirmed by Annexin V assay (HCT116: 86%; SW480: 96%). Normal cells were not altered by (PsT + NAC)® treatments.

Voacamine modulates the sensitivity to doxorubicin of resistant osteosarcoma and melanoma cells and does not induce toxicity in normal fibroblasts.

In previous studies it has been demonstrated that the plant alkaloid voacamine (1), used at noncytotoxic concentrations, enhanced the cytotoxicity of doxorubicin and exerted a chemosensitizing effect on cultured multidrug-resistant (MDR) U-2 OS-DX osteosarcoma cells. The in vitro investigations reported herein gave the following results: (i) the chemosensitizing effect of 1, in terms of drug accumulation and cell survival, was confirmed using SAOS-2-DX cells, another MDR osteosarcoma cell line; (ii) compound 1 enhanced the cytotoxic effect of doxorubicin also on the melanoma cell line Me30966, intrinsically drug resistant and P-glycoprotein-negative; (iii) at the concentrations used to sensitize tumor cells, 1 was not cytotoxic to normal cells (human fibroblasts). These findings suggest possible applications of voacamine (1) in integrative oncologic therapies against resistant tumors.

Electroporation in Translational Medicine: From Veterinary Experience to Human Oncology.

Electroporation (EP) is a broadly accepted procedure that, through the application of electric pulses with appropriate amplitudes and waveforms, promotes the delivery of anticancer molecules in various oncology therapies. EP considerably boosts the absorptivity of targeted cells to anticancer molecules of different natures, thus upgrading their effectiveness. Its use in veterinary oncology has been widely explored, and some applications, such as electrochemotherapy (ECT), are currently approved as first-line treatments for several neoplastic conditions. Other applications include irreversible electroporation and EP-based cancer vaccines. In human oncology, EP is still mostly restricted to therapies for cutaneous tumors and the palliation of cutaneous and visceral metastases of malignant tumors. Fields where veterinary experience could help smooth the clinical transition to humans include intraoperative EP, interventional medicine and cancer vaccines. This article recapitulates the state of the art of EP in veterinary and human oncology, recounting the most relevant results to date.

Innovative Codeposition of a Ag-Al2O3 Layer: An Attractive Combination of High Durability and Lack of Cytotoxicity for Public Space Applications.

Today, the use of silver in surfaces for public environments is very frequent, as it ensures high antimicrobial activities, avoiding the continuous disinfection of the surfaces themselves. Similarly, thanks to its interesting combination of technological properties, anodized aluminum is widely employed in the production of components for applications in public spaces. Therefore, this work describes a simple method of the codeposition of silver and anodized aluminum to combine the remarkable properties of Al2O3 layers with the antibacterial performances of silver. The effect of silver in modifying the durability features of the anodized aluminum layer was evaluated by means of various accelerated degradation techniques, such as the exposure in a climatic chamber to UV-B radiation or an aggressive atmosphere simulated by the Kesternich test. These analyses showed the good compatibility between Ag and the alumina matrix, whose durability performances were not particularly influenced by silver. Furthermore, the composite layers did not express relevant cytotoxicity activity, as evidenced by Trypan blue flow cytometry analysis and microscopy observations, ensuring the possible use of this material in applications in close contact with humans. This same conclusion was reached by observing an almost negligible ionic release of Ag by the composite layers, even following severe degradation of the alumina matrix due to exposure to a particular acid solution. In conclusion, this work presents an innovative material that can be used in public spaces, thanks to its interesting combination of high durability and low cytotoxicity.

'This ward has no ears': Role of the pastoral care practitioner in the hospital ward.

National Guidelines for Spiritual Care in Australia recommend incorporation of spiritual care in multidisciplinary patient care planning, however it is not known how consistently this is done. A qualitative interview study was designed to explore the practices of pastoral care practitioners in two city hospitals in Australia. Fourteen pastoral care practitioners participated (100% response rate). Interviews were taped and transcribed verbatim. Transcripts were analysed according to thematic analysis. Six themes were identified in the data. These were: (1) a vocation, (2) the role of pastoral care, (3) documentation, (4) communication with other ward staff, (5) barriers to communication, and (6) official recognition of pastoral care workers. While pastoral care workers are convinced of the importance of their work, they experience challenges in expressing this to their colleagues, which may reduce their impact on patient care. Ongoing professionalization of pastoral care will help to reduce this discrepancy.

Electrochemotherapy: An Alternative Strategy for Improving Therapy in Drug-Resistant SOLID Tumors.

Electrochemotherapy (ECT) is one of the innovative strategies to overcome the multi drug resistance (MDR) that often occurs in cancer. Resistance to anticancer drugs results from a variety of factors, such as genetic or epigenetic changes, an up-regulated outflow of drugs, and various cellular and molecular mechanisms. This technology combines the administration of chemotherapy with the application of electrical pulses, with waveforms capable of increasing drug uptake in a non-toxic and well tolerated mechanical system. ECT is used as a first-line adjuvant therapy in veterinary oncology, where it improves the efficacy of many chemotherapeutic agents by increasing their uptake into cancer cells. The chemotherapeutic agents that have been enhanced by this technique are bleomycin, cisplatin, mitomycin C, and 5-fluorouracil. After their use, a better localized control of the neoplasm has been observed. In humans, the use of ECT was initially limited to local palliative therapy for cutaneous metastases of melanoma, but phase I/II studies are currently ongoing for several histotypes of cancer, with promising results. In this review, we described the preclinical and clinical use of ECT on drug-resistant solid tumors, such as head and neck squamous cell carcinoma, breast cancer, gynecological cancer and, finally, colorectal cancer.

Complementary and Integrative Approaches to Cancer: A Pilot Survey of Attitudes and Habits among Cancer Patients in Italy.

Background: Cancer patients are among the main consumers of traditional, complementary, integrative, and alternative medicine (TCIM) such as natural products (herbals, integrators, etc.) and mind and body practices (yoga, acupuncture, etc.). Methods: A questionnaire on TCIM was submitted to 415 Italian cancer patients. The questionnaire consisted of three sections: (i) biographical and clinical information; (ii) use of natural substances; and (iii) use of mind-body practices. Results: 406 patients completed the questionnaire. The prevalence of TCIM use was 72.3%. Of them, 75.6% started to use TCIM after a tumor diagnosis. The main reasons for using TCIM were to mitigate side effects (65.0%), to regain physical and mental balance (35.9%), to relieve pain (18.3%), and to improve the efficacy of cancer therapy (16.0%). 44.7% of patients taking natural products used them during conventional therapies (chemotherapy, radiotherapy, etc.), and in 67.5% of cases without consulting a doctor. As a consequence, only about 50% of patients taking natural substances used these compounds appropriately, and the most common errors were related with the purpose of reducing the side effects of the therapy (52.3%) and for boosting immune system (32.1%). Conclusions: There is an impelling need to provide patients with scientifically validated information to raise awareness about the benefits and risks of using TCIM.

Platinum(II) chloride indenyl complexes: electrochemical and biological evaluation.

Four platinum(II) complexes of general formula [PtCl(η(1)-C(9)H(7))L(2)] [where L(2) is 1,2-bis(diphenylphosphino)ethane (dppe) 1 or cycloocta-1,5-diene (cod) 3] and [PtCl(2)L(2)] (where L(2) is dppe 2 or cod 4) were studied. Inhibition growth assays on human tumor cell lines evidenced for 1 and 3 an antiproliferative effect and, interestingly, the cytotoxic effect exerted by 1 is similar to that of cisplatin. Electrochemical and NMR measurements allowed us to determine the structural and redox properties. Investigation of the mechanism of action responsible for the cytotoxicity demonstrated a weak capacity of interacting with DNA. Some experiments performed on rat liver mitochondria indicate that 1 acts as an inducer of the mitochondrial permeability transition, thus leading to the release of proapoptotic factors, such as cytochrome c and apoptosis-inducing factor.

Role of Natural Antioxidant Products in Colorectal Cancer Disease: A Focus on a Natural Compound Derived from Prunus spinosa, Trigno Ecotype.

Colorectal cancer (CRC) is on the rise in industrialized countries, which is why it is important to find new compounds that are effective, with little or no adverse health effects. CRC arises from some cells of the epithelium which, following a series of genetic or epigenetic mutations, obtain a selective advantage. This work consists of a review on endogenous and exogenous antioxidant products that may have an efficacy in the treatment of CRC and an experimental study, in which the treatment was carried out with a natural compound with antitumor and antiproliferative activity, Prunus spinosa Trigno ecotype, patented by us, on HCT116 colorectal carcinoma cell line. The superoxide content was quantified after the treatments at different concentrations (2, 5, or 10 mg/mL) by means of the DHR123 probe; loss of the mitochondrial membrane potential with the tetramethylrodamine methyl ester (TMRM) cationic probe and reduced glutathione content (GSH) from monochlorobimane (MCB). This study revealed the importance of a careful choice of the concentration of the natural compound to be used in the CRC, due to the presence of a paradoxical effect, both antioxidant and pro-oxidant, depending on the different physiological conditions of the cell.

Electrochemotherapy with Mitomycin C Potentiates Apoptosis Death by Inhibiting Autophagy in Squamous Carcinoma Cells.

We investigated the chemosensitizing effect of electroporation (EP), which, using electrical pulses, permeabilizes cancer cells to drugs. The study involved two human hypopharyngeal and tongue carcinoma cell lines. The surface and intracytoplasmic expression of P-gp were evaluated by flow cytometry, demonstrating that both lines were intrinsically resistant. After establishing the optimal dose of mitomycin C (MMC) to be used, in combination with EP, we showed, by both MTT assay and optical and electron scanning microscopy, the potentiating cytotoxic effect of EP with MMC compared to single treatments. Flow cytometry showed that the cytotoxicity of EP + MMC was due to the induction of apoptosis. In addition to verifying the release of cytochrome C in EP + MMC samples, we performed an expression analysis of caspase-3, caspase-9, Akt, pAkt, HMGB1, LC3I, LC3II, p62, Beclin1, and associated proteins with both apoptotic and autophagic phenomena. Our results were confirmed by two veterinary patients in whom the EP + MMC combination was used to control margins after the resection of corneal squamous carcinoma. In conclusion, we affirmed that the effect for which EP enhances MMC treatment is due to the inhibition of the autophagic process induced by the drug in favor of apoptosis.

Exposure to ZnO nanoparticles induces oxidative stress and cytotoxicity in human colon carcinoma cells.

Engineered nanoparticles offer great promise in many industrial and biomedical applications, however little information is available about gastrointestinal toxicity. The purpose of this study was to assess the cytotoxicity, oxidative stress, apoptosis and proinflammatory mediator release induced by ZnO nanoparticles on human colon carcinoma LoVo cells. The biological activity of these particles was related to their physico-chemical characteristics. The physico-chemical characteristics were evaluated by analytical electron microscopy. The cytotoxicity was determined by growth curves and water-soluble tetrazolium assay. The reactive oxygen species production, cellular glutathione content, changes of mitochondrial membrane potential and apoptosis cell death were quantified by flow cytometry. The inflammatory cytokines were evaluated by enzyme-linked immunoadsorbent assay. Treatment with ZnO (5µg/cm(2) corresponding to 11.5µg/ml) for 24h induced on LoVo cells a significant decrease of cell viability, H2O2/OH increase, O2(-) and GSH decrease, depolarization of inner mitochondrial membranes, apoptosis and IL-8 release. Higher doses induced about 98% of cytotoxicity already after 24h of treatment. The experimental data show that oxidative stress may be a key route in inducing the cytotoxicity of ZnO nanoparticles in colon carcinoma cells. Moreover, the study of the relationship between toxicological effects and physico-chemical characteristics of particles suggests that surface area does not play a primary role in the cytotoxicity.

The Exploitation of Liposomes in the Inhibition of Autophagy to Defeat Drug Resistance.

Autophagy is a mechanism involved in many human diseases and in cancers can have a cytotoxic/cytostatic or protective action, being in the latter case involved in multidrug resistance. Understanding which of these roles autophagy has in cancer is thus fundamental for therapeutical decisions because it permits to optimize the therapeutical approach by activating or inhibiting autophagy according to the progression of the disease. However, a serious drawback of cancer treatment is often the scarce availability of drugs and autophagy modulators at the sites of interest. In the recent years, several nanocarriers have been developed and investigated to improve the solubility, bioavailability, controlled release of therapeutics and increase their cytotoxic effect on cancer cell. Here we have reviewed only liposomes as carriers of chemotherapeutics and autophagy inhibitors because they have low toxicity and immunogenicity and they are biodegradable and versatile. In this review after the analysis of the dual role of autophagy, of the main autophagic pathways, and of the role of autophagy in multidrug resistance, we will focus on the most effective liposomal formulations, thus highlighting the great potential of these targeting systems to defeat cancer diseases.

Voacamine: Alkaloid with its essential dimeric units to reverse tumor multidrug resistance.

Search for natural substances in association with conventional chemotherapeutic drugs with a chemiosensitizing action easily accessible to the tumor mass has encouraged our studies on voacamine (VOA) and its monomeric units, voacangine and vobasine. Our previous results showed that VOA sensitized multidrug resistant (MDR) osteosarcoma cells (U-2 OS/DX) to doxorubicin (DOX) cytotoxicity. VOA, extracted by Peschiera fuchsiaefolia plant, is a bisindole alkaloid consisting of an Iboga skeleton (voacangine) directly linked to a 2-acyl indole unit (vobasine). High-performance thin-layer chromatography densitometry demonstrated the purity of VOA, voacangine and vobasine samples. Flow cytometry analysis showed that VOA, voacangine and vobasine enhanced DOX accumulation of U-2 OS/DX cells, in equally way, whereas VOA reduced more efficiently DOX efflux. Optical microscopy and clonogenic assay confirmed that VOA was more effective than voacangine and vobasine in enhancing DOX cytotoxic effect. These results showed that monomers linked together are necessary to modulate resistant phenotype of osteosarcoma cells. To complete the study, we evaluated the effect of three compounds on microtubules by confocal microscopy, suggesting that only the whole molecule depolymerizes the microtubules blocking so DOX efflux-mediated by vesicles.

In vitro toxicity assessment of hydrogel patches obtained by cation-induced cross-linking of rod-like cellulose nanocrystals.

With the purpose of designing active patches for photodynamic therapy of melanoma, transparent and soft hydrogel membranes (HMs) have been fabricated by cation-induced gelation of rod-like cellulose nanocrystals (CNCs) bearing negatively charged carboxylic groups. Na+ , Ca2+ , Mg2+ have been used as cross-linkers of cellulose nanocrystal (CNC). The biosafety of this material and of its precursors has been evaluated in vitro in cell cultures. Morphological changes, cell organelles integrity, and cell survival with the tetrazolium salt reduction (MTT) assay were utilized as tests of cytotoxicity. Preliminary investigation was performed by addition of the hydrogel components to the cell culture medium and by incubations of the CNC-HM in direct and indirect contact with a confluent monolayer of A375 melanoma cells. Direct contact assays suffered from interference of physical stress. Careful evaluation of cytotoxicity was obtained considering the overall picture provided by microscopy and biochemical tests performed with the CNC-HM in indirect contact with two melanoma cell lines (A375, M14) and human fibroblasts. CNCs have been demonstrated to be a safe precursor material and CNC-HMs have a good biocompatibility provided that the excess of cations, in particular of Ca2+ is removed. These results indicate that CNC and can be safely used to fabricate biomedical devices such as transparent hydrogel patches, although attention must be paid to the fabrication procedure.