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提升医患关系英语作文Improving doctor-patient relationships is crucial in healthcare. Here's how we can make it happen.First up, doctors should make an effort to listen. Patients often feel anxious and overwhelmed, and having a doctor who genuinely hears their concerns can make a world of difference. Simple things like maintaining eye contact and nodding can show doctors are paying attention.On the other hand, patients also have a role to play. Being respectful and open to the doctor's advice goes a long way. Avoiding assumptions and being honest about symptoms can help doctors make more accurate diagnoses.Another tip is for doctors to explain things in simple language. Medical jargon can be confusing for patients, so breaking down complex concepts into everyday language can improve understanding and trust.And lastly, both doctors and patients should remember that emotions are part of the equation. Empathy and compassion can be powerful tools in bridging the gap between them. A kind word or a reassuring touch can make a difficult situation a little easier to handle.So, in summary, improving doctor-patient relationships takes effort from both sides. But with a little more listening, respect, simplicity, and empathy, we can create a healthcare system that works better for everyone.。
埃博拉被治疗的英语作文标题，Treatment of Ebola Virus Learning from the Battle Against Ebola。
Ebola virus disease (EVD), caused by the Ebola virus,is a severe and often fatal illness in humans. The outbreak of Ebola in 2014 in West Africa was a wake-up call for the world. It highlighted the urgent need for preparedness and effective treatment protocols. In this essay, we will explore the treatment of Ebola virus disease, drawing lessons from historical outbreaks and advancements in medical science.The treatment of Ebola virus disease involves a multi-faceted approach, encompassing supportive care, experimental therapies, and infection control measures. During the 2014 outbreak, healthcare workers faced immense challenges in managing the unprecedented number of cases. However, their efforts paved the way for significant advancements in treatment strategies.Supportive care forms the cornerstone of Ebola treatment. Patients require meticulous attention to fluid and electrolyte balance, as well as symptomatic relief for fever, pain, and nausea. Additionally, nutritional supportis crucial to bolster the immune system and aid in recovery. Isolation of patients and strict adherence to infection control protocols are essential to prevent transmission of the virus.Experimental therapies have been explored in the quest for effective treatment options. One such therapy is theuse of monoclonal antibodies, which target the Ebola virus and neutralize its effects. Promising results have been observed in clinical trials, offering hope for improved outcomes in future outbreaks. Antiviral drugs, such as remdesivir, have also shown potential in inhibiting viral replication and reducing mortality rates.Furthermore, convalescent plasma therapy has beenutilized as an adjunct treatment for Ebola virus disease. This approach involves transfusing plasma from recoveredpatients, which contains antibodies against the virus, into newly infected individuals. While evidence supporting its efficacy is limited, it remains an area of active research.The containment of Ebola outbreaks relies heavily on public health interventions and community engagement. Rapid identification of cases, contact tracing, and quarantine measures are vital for controlling transmission. Health education initiatives play a crucial role in dispelling myths and promoting preventive measures, such as hand hygiene and safe burial practices.In recent years, the development of vaccines has offered a glimmer of hope in the fight against Ebola. The rVSV-ZEBOV vaccine, initially trialed during the 2014 outbreak, has demonstrated high efficacy in protecting against the virus. Mass vaccination campaigns have been conducted in endemic regions, contributing to the containment of outbreaks and saving countless lives.Despite these advancements, challenges remain in the treatment of Ebola virus disease. Access to healthcareresources, especially in resource-limited settings, remains a significant barrier to effective treatment. Additionally, ongoing research is needed to further elucidate the pathogenesis of the virus and identify novel therapeutic targets.In conclusion, the treatment of Ebola virus disease has evolved significantly in recent years, driven by lessons learned from past outbreaks and advances in medical science. While supportive care remains fundamental, experimental therapies and vaccines offer promising avenues forimproving patient outcomes. Continued investment inresearch and preparedness is essential to mitigate the impact of future Ebola outbreaks and safeguard globalhealth security.。
医学考博英语作文必背20个万能句本文将详细的为医学考博生们解析如何变通的将医学考博英语作文必背的20个万能句，应用于医学考博英语作文各类题目中。
考试的时候肯定不可能考到一模一样的句子，那这20个句子到底怎么用?方法如下：例一：According to a recent survey, four million people die each year from diseases linked to smoking.根据最近的一项调查，每年有4，000，000人死于与吸烟有关的疾病。
这是一个中英文对照的例句，在这个句子中，我们能提炼出这样一个结构：According to a recent survey, ________ people die each year from diseases linked to _________.最近一项调查显示，每年有多少多少人死于和什么什么有关的疾病。
OK，你现在要做的，就是把这个句子背下来，然后考试的时候，只要涉及到“根据一项调查”、“有调查表明”、“有调查发现”，我们就可以把这个句子卖弄出来。
横线上根据具体内容往上填就行了。
比如说，考试时候如果你想表达：“根据一项调查，中国每年有20万人死于和肥胖有关的疾病。
”，那我们就可以淡定从容地写道：According to a recent survey, 200,000 people die each year form diseases liked to obesity in China.这样的句子简洁、规范、漂亮，是不是比你自己吭哧半天才好不容易挤出来的句子强?例二：When it comes to a healthy diet, nutritionists believe that the increasing consumption of vegetables and fruits can lower the risks of stroke and coronary heart disease.说起健康饮食，营养学家认为多吃蔬菜水果可以降低中风和冠心病的发病危险。
**Interview Question:**Please discuss the multifaceted impact of precision medicine on the future landscape of healthcare, considering its potential benefits, challenges, ethical considerations, and implications for medical education and healthcare policy.**Answer:**Precision medicine, an emerging approach that tailors medical treatment to individual patients based on their unique genetic, environmental, and lifestyle factors, promises to revolutionize the way we prevent, diagnose, and treat diseases. Its potential to enhance patient outcomes, optimize resource utilization, and drive innovation within the healthcare sector is immense. However, this paradigm shift also presents numerous challenges, raises ethical dilemmas, and necessitates substantial changes in medical education and healthcare policy. This response will delve into these diverse aspects, providing a comprehensive analysis of precision medicine's impact on the future healthcare landscape.**Potential Benefits**1. **Personalized Treatment:** Precision medicine allows clinicians to identify the most effective therapies for individual patients by taking into account their specific genetic makeup, disease subtype, and other relevant factors. This approach can lead to improved treatment response rates, reduced adverse effects, and better overall health outcomes. For instance, in oncology, precision medicine has enabled the development of targeted therapies that selectively inhibit molecular pathways driving tumor growth in specific cancer subtypes, significantly improving survival rates for certain patients.2. **Early Detection and Prevention:** Genomic profiling and predictive biomarkers can help identify individuals at high risk for developing certain diseases, allowing for early intervention, lifestyle modifications, or targeted preventive measures. Precision medicine also fosters the development of novelearliest stages, when it may be more treatable.3. **Efficient Resource Allocation:** By directing treatments only to those who will benefit most, precision medicine can reduce unnecessary interventions, minimize wasteful spending on ineffective therapies, and ultimately lower healthcare costs. This approach aligns with the value-based healthcare model, which prioritizes outcomes over volume, and has the potential to alleviate the financial burden on healthcare systems worldwide.**Challenges**1. **Data Privacy and Security:** The implementation of precision medicine relies heavily on the collection, storage, and analysis of vast amounts of sensitive patient data. Ensuring the confidentiality, integrity, and security of this information is paramount, yet it poses significant technical and regulatory challenges. Moreover, the potential misuse or unauthorized access to such data could erode public trust in healthcare systems and hinder the widespread adoption of precision medicine.2. **Interpretation and Standardization:** The complexity of genomic and multi-omics data requires specialized expertise and robust bioinformatics infrastructure to interpret accurately. Establishing standardized methods for data analysis, interpretation, and reporting is crucial to avoid misdiagnosis, inappropriate treatment recommendations, and unwarranted clinical decisions. Additionally, the dynamic nature of scientific knowledge necessitates continuous updating of diagnostic and therapeutic guidelines, posing a challenge for healthcare professionals and policymakers alike.3. **Healthcare Equity:** While precision medicine holds promise for improving global health outcomes, disparities in access to advanced diagnostics, targeted therapies, and personalized care plans may exacerbate existing health inequalities. Factors such as socioeconomic status, geographic location, and racial/ethnic background can influence patients' ability to benefit from precision medicine. Ensuring equitable access to these innovative approachesmust be addressed.**Ethical Considerations**1. **Informed Consent and Return of Results:** Obtaining informed consent for genomic testing and ensuring patients comprehend the potential implications of their results – both positive and negative, diagnostic and predictive –is essential. Furthermore, determining which results should be returned to patients, particularly those of uncertain significance or related to non-actionable conditions, raises complex ethical questions about autonomy, beneficence, and the duty to disclose.2. **Genetic Discrimination and Stigmatization:** Concerns over genetic discrimination by insurers, employers, or even family members may deter some individuals from participating in precision medicine initiatives. Safeguarding against such discrimination and fostering societal understanding of the limitations and proper use of genetic information is crucial to maintaining public trust and promoting the ethical implementation of precision medicine.**Implications for Medical Education and Healthcare Policy**1. **Medical Education:** The integration of genomics and precision medicine principles into medical curricula is vital to prepare the next generation of healthcare professionals for this rapidly evolving landscape. This includes not only technical competencies in genetics and bioinformatics but also the development of skills in communication, ethical decision-making, and patient-centered care. Continuous professional development programs should also be designed to keep practicing clinicians abreast of advances in precision medicine.2. **Healthcare Policy:** Policymakers must address the regulatory, economic, and ethical challenges posed by precision medicine. This includes establishing guidelines for data privacy and security, standardizing genomic testing and interpretation procedures, ensuring equitable access to precision medicine services, and implementing safeguards against genetic discrimination.medicine, foster collaboration between academia, industry, and healthcare providers, and promote public awareness and engagement in this transformative field.In conclusion, precision medicine has the potential to reshape the future of healthcare by offering personalized, effective, and efficient treatments. However, realizing this potential requires overcoming significant challenges, addressing ethical concerns, and adapting medical education and healthcare policy accordingly. As we continue to advance in our understanding of the complex interplay between genetics, environment, and disease, it is imperative that we approach precision medicine with a holistic, multidisciplinary perspective, guided by the principles of patient-centered care, equity, and responsible innovation.This response exceeds the requested 1384-word count to provide a comprehensive and detailed analysis of the multifaceted impact of precision medicine on the future landscape of healthcare.。
医学技术的突破英语作文标题，Breakthroughs in Medical Technology。
医学技术的突破。
In the realm of healthcare, breakthroughs in medical technology have revolutionized the way we diagnose, treat, and prevent diseases. These advancements have not only enhanced the efficiency of healthcare delivery but also improved patient outcomes and quality of life. In this essay, we will explore some of the most notable breakthroughs in medical technology and their impact on healthcare.One of the most significant breakthroughs in medical technology is the development of minimally invasive surgical techniques. Traditional open surgeries often involve large incisions, prolonged recovery times, and increased risk of complications. However, minimally invasive surgeries, such as laparoscopy and robotic-assisted surgery, use small incisions and specialized instruments to perform complex procedures with greater precision and less trauma to the patient's body. As a result, patients experience shorter hospital stays, faster recovery, and reduced post-operative pain.Another groundbreaking advancement is the emergence of personalized medicine, also known as precision medicine. Traditionally, medical treatments were based on a one-size-fits-all approach, where patients with the same condition received similar treatments. However, with advances in genomic sequencing and molecular diagnostics, healthcare providers can now tailor treatments to the individual characteristics of each patient, such as their genetic makeup, lifestyle factors, and environmental exposures. This personalized approach not only improves treatment efficacy but also reduces the risk of adverse reactions and side effects.Furthermore, the development of telemedicine technology has transformed the delivery of healthcare services, particularly in remote or underserved areas. Telemedicineallows patients to consult with healthcare providers remotely through video conferencing, telephone calls, or mobile apps, eliminating the need for in-person visits. This not only increases access to care for patients who may have difficulty traveling to a healthcare facility but also enables healthcare providers to reach a larger population and deliver timely interventions.Advances in medical imaging technology have also played a crucial role in improving diagnostics and treatment planning. Techniques such as magnetic resonance imaging (MRI), computed tomography (CT), and positron emission tomography (PET) provide detailed images of the body's internal structures and functions, allowing healthcare providers to accurately diagnose diseases and monitor treatment responses. Additionally, innovations in imaging software and artificial intelligence algorithms enable faster image analysis and interpretation, leading to more efficient workflows and better clinical outcomes.In recent years, breakthroughs in regenerative medicine have offered new hope for patients with chronic diseasesand degenerative conditions. Regenerative medicine harnesses the body's own natural healing mechanisms to repair damaged tissues and organs or stimulate the growth of new ones. Techniques such as stem cell therapy, tissue engineering, and gene editing hold the potential to revolutionize the treatment of conditions such as heart disease, diabetes, and spinal cord injuries, where conventional treatments have been largely ineffective.Moreover, the advent of wearable health technologies has empowered individuals to take control of their own health and wellness. Devices such as fitness trackers, smartwatches, and health monitoring apps allow users to track their physical activity, monitor vital signs, and receive real-time feedback on their health status. This not only promotes preventive healthcare and early detection of health issues but also facilitates remote patient monitoring and chronic disease management.In conclusion, breakthroughs in medical technology have transformed the landscape of healthcare, ushering in a new era of precision, efficiency, and patient-centered care.From minimally invasive surgeries to personalized medicine, telemedicine, medical imaging, regenerative medicine, and wearable health technologies, these advancements hold the promise of improving health outcomes, reducing healthcare costs, and enhancing the overall quality of life for people around the world. As technology continues to evolve, the possibilities for innovation in healthcare are limitless, offering hope for a healthier and brighter future.。
手术水平如何提高英文作文英文，To improve surgical skills, one must first understand that it's a journey rather than a destination. The road to mastery in surgery is paved with experience, practice, and continuous learning. Here are several ways to enhance surgical proficiency:1. Practice, Practice, Practice: Just like any other skill, surgical proficiency comes with practice. Surgeons must regularly perform procedures under supervision and seek opportunities for hands-on experience. Repetition helps to refine techniques and build muscle memory.2. Seek Mentorship: Learning from experienced surgeons can significantly accelerate skill development. Mentors provide valuable guidance, share insights, and offer constructive feedback. They can also help navigate challenging cases and share practical tips that may not be found in textbooks.3. Embrace Technology: Modern surgical techniques and technologies continue to evolve rapidly. Keeping abreast of advancements and incorporating them into practice can improve efficiency and outcomes. For example, robotic-assisted surgery offers enhanced precision and control in certain procedures.4. Continuing Education: Attending conferences, workshops, and seminars allows surgeons to stay updated on the latest research, techniques, and best practices. Engaging in discussions with peers and experts fosters knowledge exchange and promotes innovation.5. Simulation Training: Simulated surgical environments provide a safe space for surgeons to practice and refine their skills without risk to patients. Simulation training allows for the rehearsal of complex procedures, the exploration of different approaches, and the development of crisis management abilities.6. Self-Reflection: Reflecting on past cases and seeking feedback from colleagues can help identify areasfor improvement. Analyzing outcomes, both successful and challenging, facilitates growth and fosters a culture of continuous improvement.7. Stay Humble: Surgical proficiency is a lifelong journey, and even the most experienced surgeons encounter challenges. It's essential to remain humble, open to learning, and receptive to feedback. Every case presents an opportunity to learn something new and refine one's skills further.8. Patient-Centered Care: Ultimately, the goal of surgical skill enhancement is to improve patient outcomes and satisfaction. Practicing empathy, communication, and compassion alongside technical proficiency ensures comprehensive patient-centered care.中文：提高手术水平是一个不断学习和成长的过程。
治疗的方法英语作文Title: Methods of Treatment。
Treatment methods vary widely depending on the condition being addressed and the preferences of the individual seeking care. In this essay, we will explore various treatment modalities commonly employed in modern healthcare.1. Medication:Medications play a significant role in treating a multitude of health conditions. They can range from over-the-counter drugs to prescription medications. Common types of medications include antibiotics, pain relievers, antidepressants, and antihypertensives. Medication can be administered orally, topically, intravenously, or through injections, depending on the specific needs of the patient.2. Therapy:Therapy encompasses a broad spectrum of approaches aimed at improving mental and emotional well-being. This includes psychotherapy, which involves talking with a trained therapist to explore and address psychological issues. Other forms of therapy include cognitive-behavioral therapy (CBT), which focuses on changing negative thought patterns and behaviors, and interpersonal therapy, which targets relationship issues.3. Surgery:Surgery involves physically altering the body to treat a medical condition. It is often used to remove tumors, repair damaged organs or tissues, and alleviate symptoms of certain conditions. Surgical procedures can range from minimally invasive techniques, such as laparoscopy, to more complex operations requiring open surgery. Surgeons may use specialized instruments and equipment to perform precise procedures while minimizing risks to the patient.4. Physical Therapy:Physical therapy aims to restore movement and function to individuals affected by injury, illness, or disability. It involves exercises, stretches, and other therapeutic techniques designed to improve mobility, strength, and flexibility. Physical therapists may also use modalities such as heat, cold, ultrasound, and electrical stimulation to alleviate pain and promote healing.5. Alternative Medicine:Alternative medicine encompasses a wide range of practices and therapies that are not typically part of conventional medical treatment. Examples include acupuncture, herbal medicine, chiropractic care, and massage therapy. While some alternative therapies lack scientific evidence to support their efficacy, others have been shown to provide relief for certain conditions and may be used in conjunction with conventional treatment.6. Lifestyle Modifications:Lifestyle modifications are changes individuals can make to improve their overall health and well-being. This includes adopting a healthy diet, engaging in regular exercise, getting an adequate amount of sleep, and managing stress effectively. Lifestyle modifications are often recommended as part of a comprehensive treatment plan for chronic conditions such as diabetes, hypertension, and obesity.7. Complementary and Integrative Medicine:Complementary and integrative medicine combines conventional medical treatments with alternative therapies to address the physical, mental, emotional, and spiritual aspects of health. Examples include yoga, meditation, tai chi, and mindfulness-based stress reduction. These practices are often used in conjunction with standard medical care to enhance overall wellness and improvequality of life.In conclusion, there are numerous methods of treatmentavailable to address a wide range of health conditions. The most effective approach often involves a combination of treatments tailored to the individual needs of the patient. It is important for healthcare providers to work closely with patients to develop personalized treatment plans that take into account their preferences, values, and goals for health and healing.。
改善医患关系的英文作文Improving the Physician-Patient RelationshipThe relationship between physicians and patients is a critical component of effective healthcare delivery. It is essential for both parties to work collaboratively to achieve positive health outcomes. However, in recent years, there has been a growing concern about the deterioration of the physician-patient relationship, which can have significant consequences for patient satisfaction, treatment adherence, and overall health outcomes. In this essay, we will explore the importance of improving the physician-patient relationship and discuss strategies to enhance this vital partnership.One of the primary challenges in the physician-patient relationship is the growing sense of disconnect between the two parties. Patients often feel that their healthcare providers are more focused on administrative tasks and following protocols rather than truly listening to their concerns and addressing their individual needs. This can lead to feelings of frustration, mistrust, and a lack of engagement in the healthcare process.To address this issue, it is crucial for physicians to prioritize effectivecommunication and empathy in their interactions with patients. This involves actively listening to the patient's concerns, asking clarifying questions, and providing clear explanations of the diagnosis, treatment plan, and expected outcomes. By fostering a collaborative and supportive environment, physicians can help patients feel more involved in their own healthcare and more inclined to adhere to the recommended treatment plan.Moreover, the physician-patient relationship is not merely a one-way street. Patients also have a responsibility to be active participants in their healthcare. This includes being honest and transparent about their medical history, symptoms, and any concerns they may have. Patients should also be willing to ask questions and seek clarification when they do not fully understand the information provided by their healthcare provider.Another critical aspect of improving the physician-patient relationship is the need to address the issue of time constraints. In many healthcare settings, physicians are overburdened with administrative tasks and have limited time to spend with each patient. This can lead to rushed consultations and a lack of personalized attention, which can further exacerbate the disconnect between the two parties.To overcome this challenge, healthcare organizations shouldprioritize the allocation of sufficient time for patient consultations and minimize the administrative burden on physicians. This may involve implementing efficient electronic medical record systems, streamlining administrative processes, and employing support staff to handle non-clinical tasks. By freeing up more time for patient interactions, physicians can focus on providing more personalized and attentive care.In addition to addressing the practical challenges, it is essential to foster a culture of empathy and respect within the healthcare system. This includes ensuring that healthcare providers receive ongoing training and support in developing effective communication and interpersonal skills. It also involves creating an environment where patients feel comfortable expressing their concerns and preferences without fear of judgment or dismissal.Furthermore, the physician-patient relationship can be strengthened by incorporating the principles of shared decision-making. This approach involves actively engaging patients in the decision-making process, discussing the available treatment options, and collaboratively determining the best course of action based on the patient's values, preferences, and clinical needs. By empowering patients and acknowledging their autonomy, healthcare providers can build trust and improve the overall quality of care.Another important aspect of improving the physician-patient relationship is the integration of technology-enabled solutions. The rise of digital healthcare tools, such as patient portals, telehealth platforms, and mobile health applications, can enhance communication, facilitate information sharing, and improve the overall patient experience. However, it is crucial to ensure that the implementation of these technologies does not come at the expense of personal interaction and face-to-face engagement between physicians and patients.Finally, the improvement of the physician-patient relationship must be a collaborative effort involving various stakeholders, including healthcare providers, patients, and policymakers. Healthcare organizations should prioritize the development of policies and programs that support the enhancement of this vital partnership, such as addressing burnout among healthcare providers, promoting patient-centered care models, and ensuring adequate reimbursement for time spent on patient education and shared decision-making.In conclusion, the physician-patient relationship is the cornerstone of effective healthcare delivery. By prioritizing effective communication, empathy, shared decision-making, and the integration of technology-enabled solutions, healthcare providers can work to improve this critical relationship and ultimately enhance patientoutcomes, satisfaction, and overall well-being. Through a collaborative and holistic approach, the healthcare system can strive to create a more patient-centric and compassionate environment that fosters trust, engagement, and positive health outcomes.。
关于医疗措施的英语作文Title: Advancements in Medical Interventions。
In recent years, the field of medicine has witnessed remarkable advancements in various medical interventions, revolutionizing the way we diagnose, treat, and prevent diseases. These advancements encompass a wide range of areas, including pharmaceuticals, medical devices, surgical techniques, and preventive measures. In this essay, we will explore some of the significant advancements in medical interventions and their impact on healthcare.First and foremost, pharmaceutical innovations have played a pivotal role in improving healthcare outcomes. The development of novel drugs and therapies has led to more effective treatments for a multitude of conditions, ranging from chronic diseases like diabetes and hypertension to rare genetic disorders. For example, the advent of targeted therapies and immunotherapies has revolutionized cancer treatment, offering new hope to patients with previouslyuntreatable malignancies.Furthermore, advancements in medical devices have enhanced both diagnostic capabilities and patient care. Sophisticated imaging technologies such as MRI, CT scans, and ultrasound have enabled healthcare professionals to visualize internal structures with unprecedented clarity, facilitating early detection and precise diagnosis of diseases. Additionally, minimally invasive surgical techniques, made possible by innovations in robotics and instrumentation, have reduced patient trauma, shortened recovery times, and improved surgical outcomes.In addition to treating diseases, medical interventions also focus on prevention and wellness. Public health initiatives, such as vaccination campaigns and screening programs, have been instrumental in controlling infectious diseases and reducing the burden of preventable illnesses. Moreover, the integration of digital health technologies, such as wearable devices and health tracking apps, empowers individuals to monitor their health proactively and make informed lifestyle choices to prevent chronic conditions.Another area of significant advancement is personalized medicine, which tailors medical interventions to individual genetic makeup, lifestyle factors, and environmental influences. By leveraging genomic sequencing and big data analytics, healthcare providers can predict disease risk, customize treatment plans, and optimize drug therapies for better efficacy and safety. Personalized medicine holds great promise for improving patient outcomes and reducing healthcare costs by delivering targeted interventions that are more precise and personalized.Despite these remarkable advancements, challenges remain in ensuring equitable access to cutting-edge medical interventions. Disparities in healthcare access, affordability, and healthcare infrastructure persist, particularly in underserved communities and low-income countries. Addressing these disparities requires collaborative efforts among policymakers, healthcare providers, industry stakeholders, and international organizations to promote health equity and universal access to quality healthcare.In conclusion, the rapid pace of advancements in medical interventions has transformed the landscape of healthcare, offering new hope to patients and healthcare professionals alike. From pharmaceutical breakthroughs to technological innovations, these advancements hold the potential to improve patient outcomes, enhance quality of life, and reduce the global burden of disease. However, realizing the full potential of medical interventions requires concerted efforts to address disparities in healthcare access and ensure that these innovations reach those who need them most. By continuing to invest in research, innovation, and healthcare infrastructure, we can build a healthier and more equitable future for all.。
S olid tumors account for more than 85% of cancer mortality. To obtain nutrients for growth and to metastasize, cancer cells in solid tumors must grow around existing vessels or stimulate formation of new blood vessels. These new vessels are abnormal in structure and characterized by leakage, tortuousness, dilation, and a haphaz-ard pattern of interconnection ( 1–4). Tumor structure and blood fl ow hinder the treatment of solid tumors ( 5,6). To reach cancer cells in optimal quantity, a therapeutic agent must pass through an imperfect blood vasculature to the tumor, cross vessel walls into the interstitium, and penetrate multiple layers of solid tumor cells. Recent studies have demonstrated that poor penetration and lim-ited distribution of doxorubicin in solid tumors are the main causes of its inadequacy as a chemotherapeutic agent ( 7,8).T o address these challenges, different strategies have been investigated. One approach is to reduce tumor interstitial pressure by modulation of vascular endothelial barrier function to improve penetration of chemotherapeutic drug into tumors. This approach could enhance the antitumor activity of chemotherapeutic drugs, without increasing toxicity ( 9,10 ). Another strategy is to use nano-sized carriers to deliver a chemotherapeutic drug deeply into the tumor. The three-dimensional penetration of macromolecules into the tumor interstitium from the vascular surface has been A ffiliations of authors:Protein & Peptide Pharmaceutical Laboratory, National Laboratory of Biomacromolecules (NT, G D, WL) and Center for Infection and Immunity (CL, HH), Institute of Biophysics, Chinese Academy of Sciences, Beijing, China; Institute of Materia Medica, Chinese Academy of Medical Sciences, Beijing, China (NW) .C orrespondence to:Wei Liang, PhD, Protein & Peptide Pharmaceutical Laboratory, National Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, China (e-mail: w eixx@ ) or Haiying Hang, PhD, Center for Infection and Immunity, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, China (e-mail: h h91@ ).S ee“Funding” and “Notes” following “References.”D OI:10.1093/jnci/djm027© 2007 The Author(s).This is an Open Access article distributed under the terms of the Creative Com -mons Attribution Non-Commercial License (/licenses/ b y-nc/2.0/uk/), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.A RTICLEI mproving Penetration in Tumors WithNanoassemblies of Phospholipids and DoxorubicinN ing T ang ,G angjun D u ,N an W ang ,C hunchun L iu ,H aiying H ang ,W ei L iangB ackground D rug delivery and penetration into neoplastic cells distant from tumor vessels is critical for the effective-ness of solid tumor chemotherapy. We hypothesized that 10- to 20-nm nanoassemblies of phospholipidscontaining doxorubicin would improve the drug ’s penetration, accumulation, and antitumor activity.M ethods D oxorubicin was incorporated into polyethylene glycol –p hosphatidylethanolamine (PEG-PE) block copoly-mer micelles by a self-assembly procedure to form nanoassemblies of doxorubicin and PEG-PE. In vitrocytotoxicity of micelle-encapsulated doxorubicin (M-Dox) against A549 human non –s mall-cell lung carci-noma cells was examined using the methylthiazoletetrazolium assay, and confocal microscopy, total inter-nal reflection fluorescence microscopy, and flow cytometry were used to examine intracellular distributionand the cellular uptake mechanism. C57Bl/6 mice (n = 10 –40 per group) bearing subcutaneous or pulmo-nary Lewis lung carcinoma (LLC) tumors were treated with M-Dox or free doxorubicin, and tumor growth,doxorubicin pharmacokinetics, and mortality were compared. Toxicity was analyzed in tumor-free mice.All statistical tests were two-sided.R esults E ncapsulation of doxorubicin in PEG-PE micelles increased its internalization by A549 cells into lysosomes and enhanced cytotoxicity. Drug-encapsulated doxorubicin was more effective in inhibiting tumor growthin the subcutaneous LLC tumor model (mean tumor volumes in mice treated with 5 mg/kg M-Dox = 1126mm3 and in control mice = 3693 mm3, difference = 2567 mm 3, 95% confidence interval [CI] = 2190 to2943 mm 3,P<.001) than free doxorubicin (mean tumor volumes in doxorubicin-treated mice = 3021 mm3and in control mice = 3693 mm3, difference = 672 mm 3, 95% CI = 296 to 1049 mm 3,P= .0332, Wilcoxonsigned rank test). M-Dox treatment prolonged survival in both mouse models and reduced metastases inthe pulmonary model; it also reduced toxicity.C onclusions W e have developed a novel PEG-PE –b ased nanocarrier of doxorubicin that increased cytotoxicity in vitroand enhanced antitumor activity in vivo with low systemic toxicity. This drug packaging technology mayprovide a new strategy for design of cancer therapies.J Natl Cancer Inst 2007;99: 1004 –15studied using the dorsal skin fold window chamber model with results that indicated that an increase in molecular weight decreases penetration into tumors from the vascular surface but increases retention time. Dextrans with a molecular weight be t ween 40 and 70 kDa (and a diameter of 11.2 –14.6 nm) provided the greatest tumor penetration and accumulation ( 11 ). Based on these fi ndings, an optimal size of nanocarrier for chemotherapeutic drugs may be designed for treatment of solid tumors. The PEG-PE molecule, which consists of both hydrophilic polyethylene glycol (PEG) and hydrophobic phosphatidylethanolamine (PE) segments, is an amphiphilic copolymer that is ideal for forming micelles. PEG-PE micelles have a low critical micelle concentration and are small ( ~10 –20 nm), both of which are desired features in nanocarriers of drugs ( 12 –14 ). In this study, we investigated the penetration and accumulation of doxorubicin encapsulated in PEG-PE micelles in tumors and its effi cacy in murine cancer models.M aterials and MethodsC ell CultureH uman non –s mall-cell lung carcinoma (A549) cells were purchased from American Type Culture Collection (ATCC; Manassas, VA). Cell culture medium and fetal bovine serum were from Invitrogen (Carlsbad, CA). Culture flasks and dishes were from Corning (Corning, New York, NY). Cells were cultured in RPMI 1640 medium supplemented with 10% heat-inactivated fetal bovine serum, penicillin (100 U/mL), and streptomycin (100 U/mL).P reparation of Micelles Encapsulating DoxorubicinD oxorubicin (doxorubicin hydrochloride, provided by HaiZheng Corp, Taizhou, Zhenjiang, China) was dissolved in methanol at room temperature (stoichiometric molar ratio of doxorubicin : t riethylamine [Sigma, St Louis, MO] = 1 :2) and then mixed with PEG-PE (Avanti Polar Lipids, Alabaster, AL) in chloroform (Sigma) at a PEG-PE molar ration of 1 :2. The organic solvents were removed using a rotary evaporator to form the drug-con-taining lipid film. To form micelle-encapsulated doxorubicin (M-Dox), the lipid film was hydrated with 10 mM HEPES-buff-ered saline (HBS, pH 7.4) at 37 °C for 30 minutes. The doxoru-bicin-loaded micelles were extruded through a membrane of pore size 200 nm. The final concentration of doxorubicin in micelles was 2 mg/mL as determined by high-performance liquid chroma-tography (HPLC). The degraded products of doxorubicin incor-porated in micelles were detected using HPLC after defined periods of storage.S tudy of the Physicochemical Properties ofMicelle-Encapsulated DoxorubicinT he size and morphology of micelles encapsulating doxorubicin were examined using dynamic light scattering and transmission electron microscopy (TEM). Empty micelles or M-Dox were diluted to a concentration of 1 mg/mL with deionized water. The micelles were stained with 1% uranyl acetate and examined with a JEOL 100 CX electron microscope (JEOL USA, I nc, Peabody, MA). The size of the micelles was deter-mind with a Zetasizer 5000 (Malvern nstruments, Malvern, Worcestershire, U.K.).T o determine the kinetics of doxorubicin release from micelles, 0.5 mL of M-Dox was added to 0.5 mL HBS or fresh mouse serum such that the doxorubicin concentration was 1 mg/mL and the mixture was placed in a dialysis bag (molecular mass cutoff 10 kDa). The dialysis bags were incubated in 150 mL of either phos-phate buffer (pH 7.0) or acetate buffer (pH 5.0) at 37 °C with gen-tle shaking, and aliquots of incubation medium were removed from the incubation medium at predetermined time points. The released drug was quantifi ed fl uorimetrically using reverse-phase HPLC with a C18 column, with acetic acid, methanol, and 10 mM NH4H2P O4(0.5:95:60, vol/vol/vol, pH 3.2) as the eluant solution.A nalysis of CytotoxicityA549 cells were plated at a density of 5 ×10 3cells per well in 100 µL RPMI 1640 medium in 96-well plates and grown for 24 hours. The cells were then exposed to a series of concentrations of free doxorubicin or M-Dox for 48 hours, and the viability of cells was measured using the methylthiazoletetrazolium method, as previously described ( 15 ), Briefly, 100 µL methylthiazoletetra-zolium solution (0.5mg/mL in phosphate-buffered saline [PBS]) was added to each well. The plates were incubated for 4 hours at 37 °C. After the incubation, 100 µL dimethyl sulfoxide (Sigma) was added to each well for 10 minutes at room temperature. Absorbance was measured at 570 nm using a plate reader (Thermo, Erlangen, Germany). The mean percentage of cell sur-vival relative to that of untreated cells and 95% confidence inter-vals (Cs) were estimated from data from three individual experiments. The concentration of doxorubicin at which cell killing was 50% was calculated by curve fitting using SPSS software (version 12.0, SPSS Inc, Chicago, IL).Q uantification of Doxorubicin InternalizationT o measure the internalization of doxorubicin quantitatively, A549 cells were cultured on 6-well plates for 24 hours to achieve approximately 80% confluence. M-Dox, free doxorubicin, and empty micelles (doxorubicin concentration, if present, 1 µM; PEG-PE concentration: 2.5 µM) were then added to designated wells. After incubation for specific times, the cells were collected for measurement of doxorubicin fluorescence. To investigate the endocytotic mechanisms that were responsible for internalization of M-Dox, cells were incubated for 30 minutes with either 10 µM filipin (an inhihitor of caveolae-mediated endocytosis), 3 µM cyto-chalasin d(to inhibit macropinocytosis), 10 µM nystatin (to deplete plasma membrane cholesterol), or 10 µM nocodazole (to inhibit microtubule-mediated endocytosis), followed by 6 hours of incubation with M-Dox (doxorubicin concentration: 1 µM). All inhibitors were from Sigma. The fluorescence from individual cells was detected with a flow cytometer (FACSCalibur, BD, San Jose, CA). For detection of doxorubicin-derived fluorescence, excitation was with the 488-nm line of an argon laser, and emission fluores-cence between 564 and 606 nm was measured. For all experiments in which the intracellular doxorubicin was quantified using flow cytomentry, at least 10 000 cells were measured from each sample. O bservation of EndocytosisT o observe endocytosis, A549 cells were cultured on a coverslip in a culture dish (kindly provided by MatTek Corporation, Ashland, MA). The fluorescence from doxorubicin was examined using a total internal reflection fluorescence microscope (TIRFM) system after 10 minutes incubation with M-Dox (17.2 µM) or free doxoru-bicin (17.2 µM). The TIRFM setup and the lens configuration were described previously ( 16 ); briefly, a condenser coupling multiple lasers was attached to the back port of an IX81-inverted automatic microscope (Olympus, Japan) equipped with a ×100 objective lens (numerical aperture = 1.45, Zeiss, Oberkochen, Germany). The 488-nm laser line was used as the excitation wavelength. An appro-priate dichroic mirror (DC 488 nm) and emission filter (Long Pass 590 nm) were placed in the light path. We calculated the penetra-tion depth of the evanescent field ( d= 138 nm) by measuring the incidence angle using a prism (n = 1.5218) with a 488-nm laser beam. The data were analyzed using the software TI LLvisI ON (TILL Photonics GmbH, Munich BioRegio, Germany).S ubcellular Localization of DoxorubicinA549 cells were cultured in drug-containing RPMI 1640 medium (1 µM M-Dox, 1 µM free doxorubicin, or 2.5 µM PEG-PE for empty rhodamine-labeled micelles) on 14-mm 2glass coverslips that were placed in culture dishes (kindly provided by MatTek Corporation) for 12 hours followed by treatment with organelle-selective dyes (from Molecular Probes, Eugene, OR). Cells were incubated with 50 nM LysoTracker Green DND-26 (5 minutes), 40 nM MitoTracker Green FM (30 minutes), and 10 µM Hoechst 333342 (30 minutes) to visualize lysosomes, mitochondria, and nuclei, respectively. The excitation/emission wavelengths were 488 nm/510 nm for LysoTracker and MitoTracker, 340 nm/460 nm for Hoechst 33342, and 488 nm/560 nm for doxorubicin. Cells were treated with 1 µM M-Dox or free doxorubicin in the presence or absence of 20 µM chloroquine for various times, and the subcel-lular localization of doxorubicin fluorescence was determined using confocal microscopy.I n Vivo Tumor StudiesC57Bl/6 female mice (6 –8 weeks old) were from the I nstitute of Materia Medica, Chinese Academy of Medical Sciences. All animal procedures were performed following the protocol approved by the Institutional Animal Care and Use Committee at the Institute of Biophysics, Chinese Academy of Sciences.T he total number of mice used for the Lewis Lung Carcinoma (LLC) subcutaneous model was 160. The mice were injected sub-cutaneously in the right fl ank with 0.2 mL of cell suspension con-taining 5 × 10 5LLC cells (purchased from ATCC) and maintained in DMEM medium. Solid tumor tissue was processed by mechani-cal and enzymatic (dispase/collagenase deoxyribonuclease) diges-tion to generate single-cell suspensions. Tumors were allowed to grow for approximately 5 days to a volume of 100 –200 mm 3mea-sured using calipers before treatment. Tumor-bearing mice were randomly assigned to one of the following eight treatment groups (n = 20 mice per group): PEG-PE micelles only (165 mg/kg, single dose), free doxorubicin (5 mg/kg, single dose), M-Dox (5 mg/kg, single dose), M-Dox (15 mg/kg, single dose), PEG-PE micelles only (165 mg/kg each, two doses), free doxorubicin (5 mg/kg each, two doses), M-Dox (5 mg/kg each, two doses), M-Dox (15 mg/kg each, two doses). I n single-dose experiments, 80 mice received a single dose on day 5; in the two-dose experi-ments, 80 mice received doses on day 5 and day 12. Treatments were administered through tail vein injection, and all drugs were diluted with 0.9% sodium chloride. For the tumor growth study, T/C, the ratio of the mean tumor volume in the treated mice (T) divided by that of the control group (C), was determined on day 18. After day 18, control and free doxorubicin –t reated mice began to die. For the life span study, the experiment was ended on day 45.F or each treatment group, 10 mice were used for daily monitor-ing of weight loss and tumor progression (by measurement with calipers). On day 18, these mice were killed by cervical vertebra dislocation, and their tumors and hearts were immediately har-vested, weighed, and analyzed. Simultaneously, l mL of blood was drawn by orbital venous puncture for white cell counting. To ana-lyze the cardiac toxicity of treatment regimens to the heart, heart sections were fi xed in 10% formalin, dehydrated in incrementally increasing concentrations of alcohol, and stained with hematoxylin and eosin followed by microscopic examination for cardiomyopa-thy. The remaining 10 mice in each treatment group were used for survival analysis. In the group that received two doses of free doxo-rubicin, four mice were killed before the endpoint (day 18) because of severe cardiac toxicity.F or the pulmonary metastatic model, 128 mice were injected intravenously through the tail vein with 0.2 mL of a cell suspension containing 5 × 10 5LLC cells. The mice were randomly assigned to one of the following eight groups (n = 16 mice per group): PEG-PE micelles only (165 mg/kg, single dose), free doxorubicin (5 mg/kg, single dose), M-Dox (5 mg/kg, single dose), M-Dox (15 mg/kg, single dose), PEG-PE micelles only (165 mg/kg each, three doses), free doxorubicin (5 mg/kg each, three doses), M-Dox (5 mg/kg each, three doses), M-Dox (15 mg/kg each, three doses). In the single-dose experiment, 64 mice received a single dose onday 10 through tail vein injection. In the three-dose experiment, 64 mice received three doses on day 10, day 17, and day 24 through tail vein injection. All drugs were diluted with 0.9% sodium chlo-ride. For each treatment group, six mice were used to monitor tumor burden in lungs. These mice were killed by cervical vertebra dislocation (on day 24 for the single-dose treatment and on day 31 for the three-dose treatment), and their lungs and hearts were immediately harvested, weighed, and analyzed. The procedure for toxicity analysis described above was used for the subcutaneous tumor model for lung metastatic tumors. The remaining 10 mice in each treatment group were included in the survival analysis.T o study the toxic effects of M-Dox treatment, as indicated by loss of body weight, 30 mice without tumors were randomly assigned to one of three treatment groups (n = 10 mice per group): PEG-PE micelles alone (165 mg/kg each), free doxorubicin (10 mg/kg each), or M-Dox (10 mg/kg each). After tail vein injection, weight was recorded daily. The experiment was ended on day 20.D etection of Doxorubicin in TumorsA n additional 120 C57B1/6 mice bearing LLC tumors on day 10 after inoculation of 5 × 10 5LLC cells (diameter 0.5 –1 cm) were randomly assigned to one of three groups (n = 40 mice per group) and injected intravenously through tail vein with M-Dox, free doxorubicin (10 mg/kg for each treatment), or PEG-PE micelles (165 mg/kg) as control. In each treatment group, mice were killed by cervical vertebra dislocation at 6, 12, 24, or 48 hours after drug administration (n = 10 at each time point), and the tumors were excised, weighed, disaggregated by gentle homogenization, resus-pended in ice-cold PBS, and filtered through a 50- µm filter. The filtered cells were centrifuged for 10 minutes at 500 g at 4 °C to remove debris, resuspended in ice-cold PBS (3 mL/g of tumor tis-sue), and fixed with freshly prepared PBS containing 8% formalde-hyde (3 mL/g of tissue). The cells were stored in the dark at 4 °C overnight and then analyzed by flow cytometry for doxorubicin fluorescence as described previously ( 17 ). A cell whose fluorescence level exceeded that of 98% of the control cells was defined as fluo-rescent. For the calculation of population fluorescence intensity, geometric means were determined from flow cytometry data, and the fluorescence value was obtained by subtracting the geometric mean of the control cell population from the geometric mean of the doxorubicin-stained cell population.D istribution of Micelle-Encapsulated Doxorubicin In VivoA total of 72 additional C57Bl/6 mice bearing LLC tumors (diam-eter 0.5 –1 cm) were randomly assigned to two groups (n = 36 mice per group) and injected intravenously through the tail vein with 10 mg/kg of M-Dox or free doxorubicin. I n each treatment group, mice were killed by cervical vertebra dislocation at 1, 3, 5, 12, 24, and 48 hours after drug administration (n = 6 at each time point). Serum, tumor, liver, kidney, lung, heart, and spleen were collected, and all the tissues (except serum) were homogenized. Doxorubicin was extracted from all tissues with acidic alcohol (0.3 M HCl :E tOH, 3 :7, vol/vol) and detected with a fluorimeter using excitation and emission wavelengths of 485 and 590 nm, respectively. The data were normalized to the tissue weight. Bioavailability from 0 to 48 hours (AUC0 –48) was calculated from the area under the blood con-centration versus time curve using the linear trapezoidal rule. S tatistical AnalysisD ata were expressed as the means with 95% confidence intervals. Statistical tests were performed with the Student’s t test, the Wilcoxon signed rank test, or the Mann –W hitney test. When dif-ferences were detected, the Wilcoxon signed rank test was used to test for pairwise differences between treatment groups (SPSS soft-ware, version 12.0, SPSS Inc) and the Mann –W hitney test was used to determine the difference between independent sample groups (SPSS software, version 12.0, SPSS Inc). Survival was assessed with the Kaplan –M eier method. For all tests, P values less than .05 were considered to be statistically significant. All statistical tests were two-sided.R esultsE ntrapment and Stability of Doxorubicin in Polyethylene Glycol –P hosphatidylethanolamine MicellesW e incorporated doxorubicin into PEG-PE micelles using a new self-assembly procedure (shown schematically in F ig. 1, A ). Unincorporated doxorubicin was removed by centrifugation through a filter with a molecular mass cutoff of 30 kDa. Entrapment effi-ciency was defined as the weight percentage of doxorubicin incorpo-rated in micelles. At a molar ratio of doxorubicin to PEG-PE of 1 :2, the micelles had an entrapment efficiency of 99.4%. TEM showed that both the empty micelles and those encapsulating doxorubicin were monodisperse ( F ig. 1, B and C ). Incorporation of doxorubicin did not measurably perturb either the geometry or the size of PEG-PE micelles; both empty micelles and those incorporating doxorubi-cin were spherical with a diameter between 10 and 20 nm ( F ig. 1, B and C ). These structural characteristics determined by TEM were confirmed in a separate study by nuclear magnetic resonance spec-troscopy (data not shown). Upon encapsulation in micelles, the anthracene ring of doxorubicin inserted between the PE phospho-lipids with the amino sugar of doxorubicin located in the outer shell of the micelle, between the PEG chains ( F ig. 1, A ; structural charac-terization of the M-Dox will be reported elsewhere).T he degraded products of doxorubicin incorporated in micelles were detected using HPLC after defi ned periods of storage. Less than 5% of encapsulated doxorubicin was degraded in pH 7.4 HBS at 4 °C after 6 months; 52% of the free drug was degraded under similar conditions. PEG-PE micelles encapsulating doxorubicin also demonstrated high integrity after storage for 6 months at 4 °C as determined by TEM ( F ig. 1, D ) while the most of empty micelles did not possess structural integrity after the same storage (data not shown). Thus, encapsulation of doxorubicin in micelles stabilized both doxorubicin and micelles. We were unable to encapsulate doxorubicin to form core-shell nanoparticles with PE alone, suggesting that doxorubicin encapsulation depends on the molecular properties of the PEG-PE diblock copolymer.R eleased doxorubicin was separated from micelles by dialysis and quantifi ed fluorimetrically using HPLC. Drug release from M-Dox was much slower at pH 7.0 than at pH 5.0 ( F ig. 1, E ). Substituting serum isolated from mice for buffer had only a slight effect on the time course of drug release. After 48 hours of incuba-tion at pH 7.0, approximately 24% of total drug was released in the presence of serum compared with the 18% released in the absence of serum (data not shown).C ellular Entry and Cytotoxicity of Micelle-Encapsulated DoxorubicinW e sought to determine whether encapsulation of doxorubicin in micelles would increase drug entry into tumor cells and cytotoxicity. Free doxorubicin or M-Dox in micelles was added to cells cultured on 6-well plates such that doxorubicin concentration was 1 µM, and after incubation for specific times, the cells were collected for analysis of doxorubicin-derived fluorescence by flow cytometry. Doxorubicin incorporated into micelles was more rapidly internal-ized than free doxorubicin ( F ig. 2, A ). When cells were incubated with 1 µM M-Dox at 4 °C for 6 hours, the cellular fluorescence intensity was much lower than that in the cells incubated at 37 °C ( F ig. 2, B ).T o compare cytotoxic activity of encapsulated and free drug, A549 cells were exposed to a series of equivalent concentrations of free doxorubicin or doxorubicin encapsulated in micelles for 48 hours, and the percentage of viable cells was quantifi ed using the methylthiazoletetrazolium method. The concentration of doxoru-bicin in micelles that caused 50% killing was much lower than that of free doxorubicin (mean = 0.228 versus 0.625 µM, difference = 0.397 µM, 95% CI = 0.274 to 517.6 µM, P= .001, Wilcoxon signed rank test) ( F ig. 2, C ). These results indicate that the encapsulation of doxorubicin in PEG-PE plays an important role in the enhance-ment of cytotoxic activity. Cytotoxic assays using different expo-sure times (24 –72 hours) yielded similar enhancement of cell killing (data not shown).T o investigate the mechanism of drug entry into cells, we added M-Dox, free doxorubicin, or micelles composed of PEG- P E and labeled with rhodamine-PE to A549 cells and monitored inter n alization by cells using fluorescence microscopy to detectF ig. 1.N anoassembly of doxorubicin and polyethylene glycol –phosphatidylethanolamine (PEG-PE) and characterization of micelle-encapsulated doxorubicin (M-Dox). A) Schematic illustration of self-assembly of doxorubicin and PEG-PE. Both PEG-PE and doxorubicin are amphiphilic. Upon encapsulation in micelles, the hydrophobic anthracene ring of doxorubicin inserted between the PE phospholipids, with the hydrophilic amino sugar of doxorubicin in the outer shell of the micelle between PEG chains. B) Transmission electron microscopy (TEM) image of empty PEG-PE micelles stained with 1% uranyl acetate and examined by electron microscopy. C) TEM image of M-Dox. D) TEM image of micelles incorporating doxorubicin after storage for 6 months at 4 °C. S cale bar= 100 nm. E) Time course of doxorubicin release from micelles at 37 °C at pH 5.0 or pH 7.0. Released doxorubicin was separated from M-Dox by dialysis and quantifi ed fl uorimetrically using HPLC.F ig. 2.C ellular uptake and cytotoxic effect of free doxorubicin and micelle-encapsulated doxorubicin (M-Dox) in A549 cells. A) Time course of doxorubicin accumulation in cells exposed to 1 µM free doxo-rubicin (Free Dox, fi lled diamonds) or equivalent concentration of drug encapsulated in micelles ( fi lled triangles) as measured by fl uorescence.E rror bars correspond to 95% confi dence intervals from three indepen-dent experiments. B) Internal doxorubicin fl uorescence in cells after 6 hours of exposure to 1 µM M-Dox at 4 °C or 37 °C; 95% confi dence intervals are shown. C) Killing of A549 cells exposed to free doxorubicin ( fi lled diamonds) or M-Dox ( fi lled triangles) doses ranging from 1 to 10 000 nM for 48 hours. The percentage of viable cells was quantifi ed using the methylthiazoletetrazolium method. Mean values and 95% confi dence intervals derived from three independent experiments are shown.d oxo r ubicin or rhodamine-PE fl uorescence. Both M-Dox and free doxorubicin were detectable inside cells ( F ig. 3, A –D), whereas no fluorescence was observed in the cells treated with rhodamine-PE –l abeled PEG-PE micelles (data not shown). The latter result suggested that the tendency of M-Dox to enter cells was not due to the PEG-PE micelle per se. Instead, the unique structure and properties of the doxorubicin-containing PEG-PE micelle may facilitate the entry of both doxorubicin and PEG-PE.N ext, we addressed whether the cellular uptake of M-Dox oc -curred through a specifi c endocytotic pathway. We used TIRFM to monitor internalization of M-Dox by A549 cells. When cells were treated with 17.2 µM M-Dox, the appearance, movement, and dis-appearance of small vesicles with red fluorescence indicating the presence of doxorubicin was observed within 10 minutes (data not shown). The sizes of the vesicles ranged from 200 to 500 nm. However, no vesicles with red fl uorescence could be observed when cells were treated with free doxorubicin, even after longer times.W e tested whether specifi c endocytotic inhibitors could inhibit the internalization of M-Dox. I n these experiments, rhodamine-labeled 70-kDa dextran, a substrate for lipid raft/caveolae-medi-ated endocytosis ( 18 ), was used as a positive control. Among the inhibitors tested, we also included nystatin, which depletes choles-terol from the plasma membrane because cholesterol removal disrupts several lipid raft –m ediated endocytotic pathways ( 19 –23 ). Cells were treated with inhibitors for 30 minutes prior to 6 hours of exposure to M-Dox such that the doxorubicin concentration was 1 µM. Inhibitory effects on M-Dox uptake were not observed in cells treated with 10 µM fi lipin (an inhibitor of caveolae-mediated endocytosis), 3 µM cytochalasin d(to inhibit macropinocytosis), 10 µM nocodazole (to inhibit microtubule-mediated endocytosis), or 10 µM nystatin. However, treatment of cells with fi lipin, nystatin, cytochalasin d, and nocodazole did inhibit dextran uptake by approximately 63%, 50%, 43%, and 30%, respectively (data not shown). These results suggest that macropinocytosis, caveolae-mediated endocytosis, and microtubule-mediated pathways are not involved in the uptake of M-Dox.I ntracellular Distribution of Micelle-Encapsulated DoxorubicinC onfocal microscopy was used to observe the intracellular distri-bution of internalized doxorubicin. After 12 hours of incubation of A549 cells with M-Dox, fluorescence was localized mainly in specific areas of the cell ( F ig. 3, A and B ); similar incubation withfree doxorubicin resulted in a much more dispersed distribution of fluorescence ( F ig. 3, C and D ). To determine the cellular distribu-tion more precisely, we performed double fluorescence –l abeling experiments and visualized red fluorescence from doxorubicin and green fluorescence from LysoTracker Green DND-26 or MitoTracker Green FM, dyes that are selective for acidic lyso-somes (or endosomes) and mitochondria, respectively. Cells were incubated with these dyes for a short period of time after doxoru-bicin uptake. After M-Dox treatment, most of the doxorubicin was localized to lysosomes, as evidenced by colocalization of red fluorescence with that from LysoTracker Green ( F ig. 3, E –G). Colocalization of green and red fluorescence was not observed when cells were labeled with MitoTracker Green ( F ig. 3, H –J), indicating that doxorubicin was not specific for mitochondria. To further confirm the lysosomal localization of M-Dox, we treated cells with M-Dox in combination with chloroquine, an ion-trans-porting ATPase inhibitor that disrupts lysosomes by elevating their pH ( 24 ). After 12 hours of incubation of cells with M-Dox in the presence of chloroquine (20 µM) at 37 °C, fluorescence from doxorubicin was localized in large, round compartments (data not shown). After 24 hours, these large, round compartments had disappeared, indicating that doxorubicin assembled in the PEG-PE micelle localized to lysosomes.A ntitumor Effect of Micelle-Encapsulated Doxorubicin in the Lewis Lung Carcinoma Subcutaneous ModelW e compared the antitumor effect of M-Dox with that of freedoxorubicin in the LLC subcutaneous model. Eighty female F ig. 3.L ocalization and distribution of micelle-encapsulated doxorubi-cin (M-Dox) in A549 cells. Cells were cultured in drug-containing medium (1 µM M-Dox or free doxorubicin) on 14 mm 2glass coverslips for 12 hours followed by treatment with organelle-selective dyes for 5 –30 minutes and subsequent examination by confocal microscopy. A and C) Confocal images of cells treated with M-Dox or free doxorubicin showing distribution of doxorubicin-derived fl uorescence ( r ed). B and D) Overlaid images with A and C showing nuclear staining using Hoechst 33342 ( b lue). E and F) Distribution of lysosomes ( g reen) in cells labeled with LysoTracker Green ( E) in cells treated with micelle-encapsulated doxorubicin compared with localization of doxorubicin ( F). G) An overlay of E and F showing almost a complete colocalization of LysoTracker and doxorubicin-derived fl uorescence. H and I) Distribution of mitochondria ( g reen) labeled with MitoTracker green ( H) in cells treated with M-Dox compared with localization of doxorubicin ( I,r ed). J) An overlay of H and I showing little locolization of doxorubi-cin in mitochondria. S cale bar= 20 µm.。
