AUCTORES
Globalize your Research
Research | DOI: https://doi.org/10.31579/2640-1053/157
Department of Pharmacy, University College of Technology, Osmania University, Hyderabad, Telangana, India.
Department of Biochemistry, University College of Sciences, Osmania University, Hyderabad, Telangana, India.
*Corresponding Author: Ravikanth Chinthala, Department of Pharmacy, University College of Technology, Osmania University, Hyderabad, Telangana, India.
Citation: Ravikanth Chinthala, Sravani Putta, Surya S. Singh, (2023), Anticancer Activity of Curcumin Loaded Beta Carotene Nanoparticles in Cancer Cell Lines, J. Cancer Research and Cellular Therapeutics, 7(4); DOI:10.31579/2640-1053/157
Copyright: © 2023, Ravikanth Chinthala. this is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Received: 07 August 2023 | Accepted: 18 August 2023 | Published: 28 August 2023
Keywords: beta carotene; cell culture studies; curcumin; nanoparticles
Anticancer properties of curcumin are studied by many groups in the past and they suggest that the activity is poor because of the poor solubility and bioavailability of curcumin. Research also suggested that the nanoparticles can enhance the bioavailability of curcumin and thus enhance the anticancer activity. However, all the studies were done with nanoparticle matrices like poly lactide co glycolic acid, polycaprolactone, or other popularly known polymers. Solid lipid nanoparticles were also prepared to enhance the bioavailability. In our study, we focused on hybrid nanoparticles that are in between solid lipid nanoparticles and polymeric nanoparticles. We used beta-carotene as a matrix material to encapsulate curcumin. Further, we studied the anticancer activity in IMR 32 cells (Neuro Blastoma cells), MCF 7 cells (Breast cancer cells), HEK 293 cells (Human embryonic kidney cells). The curcumin-loaded beta-carotene nanoparticles showed potent activity compared to curcumin alone in all the cell lines. Thus, it can be concluded that the curcumin-loaded beta-carotene nanoparticles are a good option for studying the benefits of curcumin in cancer.
Curcumin, a natural compound found in turmeric, has been the subject of scientific research for its potential health benefits, including its effects on cancer. While more studies are needed to fully understand its mechanisms and efficacy, some potential benefits of curcumin in cancer are anti-inflammatory, antioxidant, anticancer properties, angiogenesis inhibition, etc. Studies suggest it has strong anti-inflammatory properties, which can help reduce inflammation in the body [1]. Chronic inflammation is linked to the development and progression of cancer, and curcumin may play a role in inhibiting pro-inflammatory pathways. Curcumin is a potent antioxidant, meaning it can neutralize free radicals and reduce oxidative stress. Free radicals can damage cells and DNA, leading to cancer development, and the antioxidant properties of curcumin may help prevent or slow down cancer progression. Anti-cancer activity: Curcumin has demonstrated anti-cancer effects in preclinical studies, primarily through its ability to inhibit the growth and spread of cancer cells and induce apoptosis (programmed cell death) in cancer cells [3,4]. It may also interfere with various molecular signaling pathways involved in cancer development and progression. Angiogenesis inhibition: Curcumin has been shown to inhibit angiogenesis, the process by which new blood vessels form to supply tumors with nutrients and oxygen. By
limiting angiogenesis, curcumin may hinder tumor growth and metastasis. Enhancement of chemotherapy and radiation therapy: Some studies suggest that curcumin can sensitize cancer cells to chemotherapy and radiation, making cancer treatments more effective. It may also protect normal cells from the harmful effects of these treatments. Prevention and treatment of cancer-related side effects: Curcumin's anti-inflammatory properties may help alleviate some cancer-related symptoms, such as pain, fatigue, and loss of appetite. It may also mitigate treatment side effects and improve the overall quality of life for cancer patients [5-8].
While the potential benefits of curcumin in cancer are promising, it's essential to note that research is still ongoing, and curcumin is not a replacement for conventional cancer treatments. It should be used as a complementary approach alongside standard medical therapies. Additionally, curcumin's bioavailability is limited, meaning it may not be efficiently absorbed by the body [9]. Researchers are exploring different formulations and delivery methods to improve their effectiveness. We have explored the nanoparticulate approach to the improvement in bioavailability and thus its anticancer property.
Curcumin and beta carotene were purchased from Yucca Enterprises, India, and Hefei TNJ Chemical Industries Co., Ltd, China, respectively. Ethyl acetate, EDTA, sucrose, mannitol, dextrose, trehalose, and polyvinyl alcohol were procured from Sigma-Aldrich. Purified reverse osmosis water was used for all the experiments.
Analytical method to estimate curcuminby UV-Visible spectrophotometer
To determine the wavelength at which curcumin demonstrates its highest level of absorption (λmax), a stock solution of 100 µg/mL was prepared by diluting 10 mg of curcumin in 100 mL of methanol. This solution was then analyzed using a spectrophotometer within the wavelength range of 800 to 400 nm, with methanol serving as the blank. The absorption curve revealed that the unique absorption maxima of the drug occurred at approximately 421 nm.
To create the calibration curve, a series of serial dilutions were performed starting from the stock solution of 100 µg/mL. The standards used for these dilutions ranged from 1 to 6 µg/mL.
Preparation of curcumin-loaded beta-carotene nanoparticles
The emulsion-diffusion-evaporation method was adapted to produce nanoparticles (NPs) of beta carotene. Beta carotene was dissolved in ethyl acetate due to its highly favorable solubility in this solvent, which is crucial for emulsification-based NP development. To prepare the NPs, the required amount of beta carotene was first dissolved in dichloromethane or chloroform and then emulsified in a stabilizer solution containing poly(vinyl alcohol), a commonly used nanoparticle preparation agent. Subsequently, this emulsion was diluted with water to facilitate the diffusion of the organic solvent, leading to the formation of a nanosuspension [10].
In brief, a mixture of 2.5 mL of dichloromethane or chloroform was stirred at 1200 rpm for 30 minutes until curcumin, beta-carotene, and the polymer fully dissolved (as shown in Table 3). This organic phase was then slowly added drop-by-drop into an aqueous phase containing a stabilizer to form an emulsion. To minimize droplet size, the emulsion was homogenized using a Polytron PT4000 (Polytron Kinematica, Switzerland) after one hour of agitation. Afterward, a significant volume of water was added to the emulsion to dilute it, resulting in the diffusion of the organic solvent and nanoprecipitation. Curcumin loading of 10 mg and 20 mg were used for every 50 mg of beta-carotene, as indicated in Table 3.
Freeze drying of nanoparticles
For freeze-drying, an automated system (VirTis, AdVantage, USA) previously customized for curcumin nanoparticles (NPs) was utilized. The freeze-drying process maintained a condenser temperature of 60°C and a pressure of 200 Torr throughout each stage. The nanoparticle suspension (2 ml) was introduced into glass vials with a capacity of 5 ml. The freeze-drying technique was optimized to achieve NPs with excellent dispersibility. To obtain a free-flowing and easily dispersible powder, different cryoprotectants and their concentrations were evaluated. Specifically, mannitol, sucrose, and trehalose were used as cryoprotectants at a 5% concentration relative to the NPs. After freeze-drying, the samples were reconstituted in demineralized water, and a series of tests were conducted to assess their size, drug content, surface charge, and morphology.
Characterization of nanoparticles
Zetasizer nanoZS (Malvern, UK) and the Malvern PCS software version 6.20 were used to perform Photon Correlation Spectroscopy (PCS) measurements of particle size and polydispersity index. Zetasizer nanoZS was used to measurethe zeta potentialin electrophoretic light scattering (ELS) mode. The device was kept at 25oC while the data was being collected. The stated values were the average of three different measurements.
Loading capacity and percentage yield
The weight of the freshly created formulation equal to one unit dose was taken and diluted with chloroform up to 10 mL. The resulting suspension was vortexed for one hour and then centrifuged at 6,000 rpm for 45 minutes (5804R, Eppendorf, Germany). A 0.2-m filter was used to separate and filter the supernatant. The filtrate was diluted with chloroform before being subjected to a UV spectrophotometer analysis at 421nm (Systronics 2203 Smart, India). The analysis was conducted using a UV calibration curve that was created at the same wavelength. The formulations made without curcumin were handled similarly and served as a control for the measurements. The experiment was performed 3 times using various preparations. The formula was used to determine encapsulation effectiveness.
In vitro cell culture studies
In vitro, cytotoxicity assays are commonly used for the evaluation of biomaterials / medical devices. “Cytotoxicity can be determined by evaluation of cell morphology, cell damage,and cell growth or by measurement of cellular activity. 3- [4, 5-dimethylthiazol-2-yl]-2, 5- diphenyl tetrazolium bromide” (MTT) assay is one of the methods strongly recommended by ISO 10993-5 [11]. IMR 32 cells (Neuro Blastoma cells), MCF 7 cells (Breast cancer cells), HEK 293 cells (Human embryonic kidney cells), were all kept as monolayer cultures in a 25 sq. cm flask using DMEM media that was augmented with 2% FCS and some antibiotics such as kanamycin, Penicillin, and Streptomycin. The cells were cultivated in an incubator containing 5% CO2 and maintained at 37ºC until they reached a confluence of 70%. The cells were then washed with 1x PBS, and 200 μL of 0.2% (Trypsin-EDTA) solution was added. The mixture was then allowed to incubate at room temperature for 5 minutes. The cells were then suspended in 5 ml of complete media, and 100 μL was subsequently transferred to 96 well ELISA plates. 48 hours were allotted for the growth of the cells (Table 1).
After removing the old medium from the ELISA plates containing the IMR 32 cells, MCF 7 cells, and HEK 293 cells and adding 100 µL of the fresh medium, the plates were examined under a microscope for signs of contamination. Cells were treated with seven distinct doses of undiluted test materials in triplicate, each time. Place the plate in an incubator with a humidity of 5% and 37 oC for a whole day. The plates were frequently removed for microscopic analysis to look for visible indicators of toxicity in response to the test ingredients and the controls. These visual signs include a change in the size or appearance of cellular components or a disturbance in their configuration. After a treatment of 24 hours, 20 µL of MTT solution (10 mg per mL of stock) was added to each well, and the plates were placed in an incubator at 37oC for four hours. The medium was discarded, and the formazancrystal was dissolvedin an isopropanol solution that included 0.04N HCl and observed an OD of 570 nm.
All the samples were dissolved in 100 % DMSO. Concentrations of curcumin or beta carotene or nanoparticles ranging from 12.5 µg to 150 µg were utilized. These amounts correspond to 25, 50, 75, 100, and 125respectively have been used for the MTT assay. The study was conducted in a single assay with a triplicate well.
| Formulation | 1 | 2 | 3 | 4 | 5 | 6 | 7 |
| Curcumin (µg) | 12.5 | 25 | 50 | 75 | 100 | 125 | 150 |
| Beta-carotene (µg) | 12.5 | 25 | 50 | 75 | 100 | 125 | 150 |
| Nanoparticles (µg) | 12.5 | 25 | 50 | 75 | 100 | 125 | 150 |
| Control (DMSO) (µl) | 10 | 10 | 10 | 10 | 10 | 10 | 10 |
Table 1: MTT assay concentrations
Analytical method
Figure 1: UV Calibration curve of curcumin
To determine the wavelength at which curcumin exhibits the highest level of absorption, known as λ max, a stock solution of 100 µg/ml was made by diluting 10mg of the substance in 100 ml of methanol. Using a spectrophotometer, this solution was analyzed within the wavelength range of 800 to 400 nm against methanol, which served as a blank. The drug's unique absorption maxima were found to occur around 421 nm along the absorption curve. The calibration curve of curcuminwas plotted in methanol at 421nm and a straight line was obtained. The equation obtained was y= 0.165x-0.0007 and the value of R2 was 0.999. The absorbance values are shown in Table 5 and the standard graph is shown in Figure 1.
Development of blank and curcumin-loaded beta carotene nanoparticles
All the developed nanoparticles showed particle sizes less than 200 nm (Table 2). Blank nanoparticles showed lower sizes than the drug-loaded nanoparticles. Nanoparticles showed almost similar size to the blank nanoparticles suggesting the compact nature of the beta carotene nanoparticles. The zeta potential is negative in all cases and the entrapment efficiency of curcumin resulted to be over 90%.
Freeze drying of developed nanoparticles
The freeze-dried nanoparticles retained properties similar to the initial nanoparticles with trehalose. The size seems to be slightly increased and the zeta potential decreased slightly after the freeze-drying process (Table 2) (Figure 1). The particles seem to be spherical and the size distribution seemed to be good in all cases. In particles prepared with beta carotene, there is a small peak of particles indicating some of the particles are very small in comparison to the other particles. This could be due to the lipid agglomeration that mighthave happened during the preparation process but not the freeze-drying process. The freeze-dried particles retained almost all the characteristics of the initial nanoparticles and are easily redispersible. For all the remaining studies freeze-dried nanoparticles were used.Before using them, they were kept at -80 degrees.
Table 2: Characteristics of nanoparticles
Figure 2: SEM image of nanoparticles after freeze-drying
In vitro cell culture studies
Table 3: Cell Viability Percentage in IMR32,MCF-7, HEK293 Cells
Cytotoxicity assay results show 53
The use of nanoparticles as drug delivery systems has gained significant attention in cancer research. In our study, curcumin-loaded beta-carotene nanoparticles were successfully prepared and characterized. We found that these nanoparticles were less than 200nm and possess negative zeta potential. The curcumin nanoparticles exhibited cytotoxicity against cancer cell lines, indicating their potential to enhance the anticancer activity of the loaded curcumin. The ability of nanoparticles to improve drug delivery and increase the effectiveness of therapeutic agents is attributed to their small size, which allows them to penetrate tumor tissues more efficiently. Beta carotene, a natural compound found in various fruits and vegetables, has been studied for its potential as a carrier for drug delivery systems. Its favorable characteristics, such as biocompatibility and stability, contribute to its effectiveness in improving the bioavailability of curcumin and the reason for the higher potency of nanoparticles in cytotoxicity to cancer cells.
Clearly Auctoresonline and particularly Psychology and Mental Health Care Journal is dedicated to improving health care services for individuals and populations. The editorial boards' ability to efficiently recognize and share the global importance of health literacy with a variety of stakeholders. Auctoresonline publishing platform can be used to facilitate of optimal client-based services and should be added to health care professionals' repertoire of evidence-based health care resources.
Journal of Clinical Cardiology and Cardiovascular Intervention The submission and review process was adequate. However I think that the publication total value should have been enlightened in early fases. Thank you for all.
Journal of Women Health Care and Issues By the present mail, I want to say thank to you and tour colleagues for facilitating my published article. Specially thank you for the peer review process, support from the editorial office. I appreciate positively the quality of your journal.
Journal of Clinical Research and Reports I would be very delighted to submit my testimonial regarding the reviewer board and the editorial office. The reviewer board were accurate and helpful regarding any modifications for my manuscript. And the editorial office were very helpful and supportive in contacting and monitoring with any update and offering help. It was my pleasure to contribute with your promising Journal and I am looking forward for more collaboration.
We would like to thank the Journal of Thoracic Disease and Cardiothoracic Surgery because of the services they provided us for our articles. The peer-review process was done in a very excellent time manner, and the opinions of the reviewers helped us to improve our manuscript further. The editorial office had an outstanding correspondence with us and guided us in many ways. During a hard time of the pandemic that is affecting every one of us tremendously, the editorial office helped us make everything easier for publishing scientific work. Hope for a more scientific relationship with your Journal.
The peer-review process which consisted high quality queries on the paper. I did answer six reviewers’ questions and comments before the paper was accepted. The support from the editorial office is excellent.
Journal of Neuroscience and Neurological Surgery. I had the experience of publishing a research article recently. The whole process was simple from submission to publication. The reviewers made specific and valuable recommendations and corrections that improved the quality of my publication. I strongly recommend this Journal.
Dr. Katarzyna Byczkowska My testimonial covering: "The peer review process is quick and effective. The support from the editorial office is very professional and friendly. Quality of the Clinical Cardiology and Cardiovascular Interventions is scientific and publishes ground-breaking research on cardiology that is useful for other professionals in the field.
Thank you most sincerely, with regard to the support you have given in relation to the reviewing process and the processing of my article entitled "Large Cell Neuroendocrine Carcinoma of The Prostate Gland: A Review and Update" for publication in your esteemed Journal, Journal of Cancer Research and Cellular Therapeutics". The editorial team has been very supportive.
Testimony of Journal of Clinical Otorhinolaryngology: work with your Reviews has been a educational and constructive experience. The editorial office were very helpful and supportive. It was a pleasure to contribute to your Journal.
Dr. Bernard Terkimbi Utoo, I am happy to publish my scientific work in Journal of Women Health Care and Issues (JWHCI). The manuscript submission was seamless and peer review process was top notch. I was amazed that 4 reviewers worked on the manuscript which made it a highly technical, standard and excellent quality paper. I appreciate the format and consideration for the APC as well as the speed of publication. It is my pleasure to continue with this scientific relationship with the esteem JWHCI.
This is an acknowledgment for peer reviewers, editorial board of Journal of Clinical Research and Reports. They show a lot of consideration for us as publishers for our research article “Evaluation of the different factors associated with side effects of COVID-19 vaccination on medical students, Mutah university, Al-Karak, Jordan”, in a very professional and easy way. This journal is one of outstanding medical journal.
Dear Hao Jiang, to Journal of Nutrition and Food Processing We greatly appreciate the efficient, professional and rapid processing of our paper by your team. If there is anything else we should do, please do not hesitate to let us know. On behalf of my co-authors, we would like to express our great appreciation to editor and reviewers.
As an author who has recently published in the journal "Brain and Neurological Disorders". I am delighted to provide a testimonial on the peer review process, editorial office support, and the overall quality of the journal. The peer review process at Brain and Neurological Disorders is rigorous and meticulous, ensuring that only high-quality, evidence-based research is published. The reviewers are experts in their fields, and their comments and suggestions were constructive and helped improve the quality of my manuscript. The review process was timely and efficient, with clear communication from the editorial office at each stage. The support from the editorial office was exceptional throughout the entire process. The editorial staff was responsive, professional, and always willing to help. They provided valuable guidance on formatting, structure, and ethical considerations, making the submission process seamless. Moreover, they kept me informed about the status of my manuscript and provided timely updates, which made the process less stressful. The journal Brain and Neurological Disorders is of the highest quality, with a strong focus on publishing cutting-edge research in the field of neurology. The articles published in this journal are well-researched, rigorously peer-reviewed, and written by experts in the field. The journal maintains high standards, ensuring that readers are provided with the most up-to-date and reliable information on brain and neurological disorders. In conclusion, I had a wonderful experience publishing in Brain and Neurological Disorders. The peer review process was thorough, the editorial office provided exceptional support, and the journal's quality is second to none. I would highly recommend this journal to any researcher working in the field of neurology and brain disorders.
Dear Agrippa Hilda, Journal of Neuroscience and Neurological Surgery, Editorial Coordinator, I trust this message finds you well. I want to extend my appreciation for considering my article for publication in your esteemed journal. I am pleased to provide a testimonial regarding the peer review process and the support received from your editorial office. The peer review process for my paper was carried out in a highly professional and thorough manner. The feedback and comments provided by the authors were constructive and very useful in improving the quality of the manuscript. This rigorous assessment process undoubtedly contributes to the high standards maintained by your journal.
International Journal of Clinical Case Reports and Reviews. I strongly recommend to consider submitting your work to this high-quality journal. The support and availability of the Editorial staff is outstanding and the review process was both efficient and rigorous.
Thank you very much for publishing my Research Article titled “Comparing Treatment Outcome Of Allergic Rhinitis Patients After Using Fluticasone Nasal Spray And Nasal Douching" in the Journal of Clinical Otorhinolaryngology. As Medical Professionals we are immensely benefited from study of various informative Articles and Papers published in this high quality Journal. I look forward to enriching my knowledge by regular study of the Journal and contribute my future work in the field of ENT through the Journal for use by the medical fraternity. The support from the Editorial office was excellent and very prompt. I also welcome the comments received from the readers of my Research Article.
Dear Erica Kelsey, Editorial Coordinator of Cancer Research and Cellular Therapeutics Our team is very satisfied with the processing of our paper by your journal. That was fast, efficient, rigorous, but without unnecessary complications. We appreciated the very short time between the submission of the paper and its publication on line on your site.
I am very glad to say that the peer review process is very successful and fast and support from the Editorial Office. Therefore, I would like to continue our scientific relationship for a long time. And I especially thank you for your kindly attention towards my article. Have a good day!
"We recently published an article entitled “Influence of beta-Cyclodextrins upon the Degradation of Carbofuran Derivatives under Alkaline Conditions" in the Journal of “Pesticides and Biofertilizers” to show that the cyclodextrins protect the carbamates increasing their half-life time in the presence of basic conditions This will be very helpful to understand carbofuran behaviour in the analytical, agro-environmental and food areas. We greatly appreciated the interaction with the editor and the editorial team; we were particularly well accompanied during the course of the revision process, since all various steps towards publication were short and without delay".
I would like to express my gratitude towards you process of article review and submission. I found this to be very fair and expedient. Your follow up has been excellent. I have many publications in national and international journal and your process has been one of the best so far. Keep up the great work.
We are grateful for this opportunity to provide a glowing recommendation to the Journal of Psychiatry and Psychotherapy. We found that the editorial team were very supportive, helpful, kept us abreast of timelines and over all very professional in nature. The peer review process was rigorous, efficient and constructive that really enhanced our article submission. The experience with this journal remains one of our best ever and we look forward to providing future submissions in the near future.
I am very pleased to serve as EBM of the journal, I hope many years of my experience in stem cells can help the journal from one way or another. As we know, stem cells hold great potential for regenerative medicine, which are mostly used to promote the repair response of diseased, dysfunctional or injured tissue using stem cells or their derivatives. I think Stem Cell Research and Therapeutics International is a great platform to publish and share the understanding towards the biology and translational or clinical application of stem cells.
I would like to give my testimony in the support I have got by the peer review process and to support the editorial office where they were of asset to support young author like me to be encouraged to publish their work in your respected journal and globalize and share knowledge across the globe. I really give my great gratitude to your journal and the peer review including the editorial office.
I am delighted to publish our manuscript entitled "A Perspective on Cocaine Induced Stroke - Its Mechanisms and Management" in the Journal of Neuroscience and Neurological Surgery. The peer review process, support from the editorial office, and quality of the journal are excellent. The manuscripts published are of high quality and of excellent scientific value. I recommend this journal very much to colleagues.
Dr.Tania Muñoz, My experience as researcher and author of a review article in The Journal Clinical Cardiology and Interventions has been very enriching and stimulating. The editorial team is excellent, performs its work with absolute responsibility and delivery. They are proactive, dynamic and receptive to all proposals. Supporting at all times the vast universe of authors who choose them as an option for publication. The team of review specialists, members of the editorial board, are brilliant professionals, with remarkable performance in medical research and scientific methodology. Together they form a frontline team that consolidates the JCCI as a magnificent option for the publication and review of high-level medical articles and broad collective interest. I am honored to be able to share my review article and open to receive all your comments.
“The peer review process of JPMHC is quick and effective. Authors are benefited by good and professional reviewers with huge experience in the field of psychology and mental health. The support from the editorial office is very professional. People to contact to are friendly and happy to help and assist any query authors might have. Quality of the Journal is scientific and publishes ground-breaking research on mental health that is useful for other professionals in the field”.
Dear editorial department: On behalf of our team, I hereby certify the reliability and superiority of the International Journal of Clinical Case Reports and Reviews in the peer review process, editorial support, and journal quality. Firstly, the peer review process of the International Journal of Clinical Case Reports and Reviews is rigorous, fair, transparent, fast, and of high quality. The editorial department invites experts from relevant fields as anonymous reviewers to review all submitted manuscripts. These experts have rich academic backgrounds and experience, and can accurately evaluate the academic quality, originality, and suitability of manuscripts. The editorial department is committed to ensuring the rigor of the peer review process, while also making every effort to ensure a fast review cycle to meet the needs of authors and the academic community. Secondly, the editorial team of the International Journal of Clinical Case Reports and Reviews is composed of a group of senior scholars and professionals with rich experience and professional knowledge in related fields. The editorial department is committed to assisting authors in improving their manuscripts, ensuring their academic accuracy, clarity, and completeness. Editors actively collaborate with authors, providing useful suggestions and feedback to promote the improvement and development of the manuscript. We believe that the support of the editorial department is one of the key factors in ensuring the quality of the journal. Finally, the International Journal of Clinical Case Reports and Reviews is renowned for its high- quality articles and strict academic standards. The editorial department is committed to publishing innovative and academically valuable research results to promote the development and progress of related fields. The International Journal of Clinical Case Reports and Reviews is reasonably priced and ensures excellent service and quality ratio, allowing authors to obtain high-level academic publishing opportunities in an affordable manner. I hereby solemnly declare that the International Journal of Clinical Case Reports and Reviews has a high level of credibility and superiority in terms of peer review process, editorial support, reasonable fees, and journal quality. Sincerely, Rui Tao.
Clinical Cardiology and Cardiovascular Interventions I testity the covering of the peer review process, support from the editorial office, and quality of the journal.
