Name: preparation and characterization of individual and multi-drug loaded physically entrapped polymeric micelles
Uploaded: 2015-08-28T13:00:00.000+00:00
Duration: 7 min 32 s
Description: Watch this Scientific Journal Video about Preparation and Characterization of Individual and Multi-drug Loaded Physically Entrapped Polymeric Micelles at JoVE.com

This study was supported by the grant from AACP New Pharmacy Faculty Research Award Program, Medical Research Foundation of Oregon New Investigator Grant, Oregon State University-Startup fund, and Pacific University, School of Pharmacy Start-up fund.

1. הכנת Micelles הטעון פרט ורב-תרופה על ידי שיטת יציקת ממס <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> לשקול את מ&quot;ג DTX 1 מ&quot;ג או EVR 1 או שני תרופות בכל 1 מ&quot;ג למיצלות התרופה הכפולה (DDM). </li><li style=";text-align:right;direction:rtl"> לשקול את 15 מ&quot;ג של PEG 2000 - ב -PLA 1800 או PEG 4000 - ב -PLA 2200 טוב לאו בודד או DDM. </li><li style=";text-align:right;direction:rtl"> ממיסים את התרופה / תרופות והפולימר ב 0.5 מיליליטר של אצטוניטריל ומקום בבקבוק עגול תחתון 5 מיליליטר. </li><li style=";text-align:right;direction:rtl"> טופס סרט פולימר תרופה המופצת דק על ידי מתאדה תרופת הפתרון (ים) -polymer אצטוניטריל תחת לחץ מופחת באמצעות מאייד סיבובי. הגדר את המאייד הסיבובי 100 סל&quot;ד, טמפרטורת מים באמבטיה של 40 o C ולחץ ואקום של 260 mbar דקות 5 ואחריו ירידה ל -100 mbar 3 דקות יותר. </li><li style=";text-align:right;direction:rtl"> רעננות סרט סמים-פולימר עם 0.5 מיליליטר של מים ללא יונים ב 50 o C ועדינות לנער את הבקבוק כדי ליצור את מיצלות. </li><li style=";text-align:right;direction:rtl"> סנן את התוצאהing פתרון micellar דרך מסנן ניילון 0.2 מיקרומטר כדי להסיר כל תרופה בלתי מומס או מזהמים לתוך צינור 1.5 מיליליטר צנטריפוגות. </li></ol> 2. הערכה של טעינת סמים ויציבות בMicelles שימוש ההפוך-השלב High Performance Liquid Chromatography (RP-HPLC) <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> ביצוע ניתוח RP-HPLC עם טור C8 equilibriated ב 40 מעלות צלזיוס במצב isocratic עם שלב נייד של אצטוניטריל / מים (62/38) המכיל 0.1% חומצה זרחתית ומתנול 1% בקצב זרימה של 1 מיליליטר / דקה ו הזרקת נפח של 10 μl. </li><li style=";text-align:right;direction:rtl"> לדלל מיצלות מוכנה טרי (סעיף 1) 1: 100 בשלב נייד לפני הניתוח על ידי RP-HPLC לקבוע טעינת תרופה ראשונית. מיצלות החנות חי בודדת וDDM בטמפרטורת חדר (C 25 O) במשך 48 שעות ולהכין 1 טרי: 100 דגימות מדוללות בשלב נייד כדי להעריך מחדש על ידי RP-HPLC ולקבוע סמים יציבות (ים) במיצלות מעל 24 שעות. </li><li style=";text-align:right;direction:rtl"> צג פסגות DTX וEVR ב227 ו -279 בהתאמה ננומטרעם זמני שמירה של 1.7 ו -5.7 בהתאמה דקות. לבצע את כל המדידות בשלושה עותקים. נתוני הווה כטעינה ממוצע ± SD סמים. </li></ol> 3. הערכה של גודל החלקיקים micelle ידי דינמי פיזור אור (DLS) <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> לדלל מיצלות מוכנה טרי (כמתואר בסעיף 1) במים ללא יונים ביחס של 1:20 להניב ריכוז פולימר סופי של 1.5 מ&quot;ג / מיליליטר. </li><li style=";text-align:right;direction:rtl"> מדוד את עוצמת הלייזר הוא Ne-(633 ננומטר) ב173 מעלות כדי לקבוע פיזור. לבצע את כל המדידות של 25 מעלות צלזיוס הבאות מראש איזון למשך 2 דקות. </li><li style=";text-align:right;direction:rtl"> לבצע את כל המדידות בשלושה עותקים. נתוני הווה כגודל ± SD הממוצע Z-ממוצע יחד עם מדד polydispersity (PDI) של ההפצה. </li></ol> 4. הערכת במבחנה שחרור תרופה מMicelles הפרט וDDM <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> הכן מיצלות וDDM בודדים כפי שתואר בסעיף 1. טען 2.5 מיליליטר של מיצלות לקלטת 3 מיליליטר דיאליזה עםלחתוך את משקל מולקולרי (MWCO) של 7,000 g / mol. הערה: זה MWCO נבחר כדי לאפשר את התרופה ללא תשלום (ים) יחד עם מולקולות פולימר בלתי-משויכות לפזר בחופשיות מתוך הקלטת ובכך להבטיח תנאי כיור. </li><li style=";text-align:right;direction:rtl"> מניחים את הקלטות ב2.5 L של 10 מ&quot;מ 7.4 חיץ pH פוספט (שהוכן על ידי דילול פתרון מ&quot;מ מניית 200) ולשנות את החיץ כל 3 שעות כדי להבטיח תנאי כיור. לשמור על הטמפרטורה של המאגר על 37 מעלות צלזיוס לאורך כל תקופת הניסוי. </li><li style=";text-align:right;direction:rtl"> ב 0, 0.5, 1, 2, 3, 6, 9, 12, 24, ו -48 שעות, לסגת 150 μl של הפתרון בקלטות ולהחליף עם 150 μl של חיץ טרי. </li><li style=";text-align:right;direction:rtl"> לנתח דגימות באמצעות RP-HPLC כפי שנקבע בסעיף 2 כדי לקבוע את ריכוז התרופה. עקומה-להתאים את נתוני שחרור תרופה (ים) המבוססים על מודל דיפוזיה פשוט עם עמותת מעריכי שלב אחד באמצעות תוכנת stastitical. </li><li style=";text-align:right;direction:rtl"> לחשב את הזמן הדרוש כדי להגיע ל -50% משחרור תרופה (t 1/2) של דוארתרופת אח במיצלות או DDM הפרטניים המבוסס על העקומה ההולמת. לבצע את כל המדידות ברביעייה. </li></ol>

<ol>
	<li>Yokoyama, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Polymeric+micelles+as+a+new+drug+carrier+system+and+their+required+considerations+for+clinical+trials.">Polymeric micelles as a new drug carrier system and their required considerations for clinical trials.</a> Expert Opin Drug Deliv. 7, 145-158 (2010).</li><li>Shin, H. C., Alani, A. W., Rao, D. A., Rockich, N. C., Kwon, G. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multi-drug+loaded+polymeric+micelles+for+simultaneous+delivery+of+poorly+soluble+anticancer+drugs.">Multi-drug loaded polymeric micelles for simultaneous delivery of poorly soluble anticancer drugs.</a> J Control Release. 140, 294-300 (2009).</li><li>Adams, M. L., Lavasanifar, A., Kwon, G. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Amphiphilic+block+copolymers+for+drug+delivery.">Amphiphilic block copolymers for drug delivery.</a> J Pharm Sci. 92, 1343-1355 (2003).</li><li>Oerlemans, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Polymeric+micelles+in+anticancer+therapy:+targeting,+imaging+and+triggered+release.">Polymeric micelles in anticancer therapy: targeting, imaging and triggered release.</a> Pharm Res. 27, 2569-2589 (2010).</li><li>Shin, H. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+3-in-1+polymeric+micelle+nanocontainer+for+poorly+water-soluble+drugs.">A 3-in-1 polymeric micelle nanocontainer for poorly water-soluble drugs.</a> Mol Pharm. 8, 1257-1265 (2011).</li><li>Hasenstein, J. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antitumor+activity+of+Triolimus:+a+novel+multidrug-loaded+micelle+containing+Paclitaxel+Rapamycin,+and+17-AAG.">Antitumor activity of Triolimus: a novel multidrug-loaded micelle containing Paclitaxel Rapamycin, and 17-AAG.</a> Mol Cancer Ther. 11, 2233-2242 (2012).</li><li>Mazzaferro, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bivalent+sequential+binding+of+docetaxel+to+methyl-beta-cyclodextrin.">Bivalent sequential binding of docetaxel to methyl-beta-cyclodextrin.</a> Int J Pharm. 416, 171-180 (2011).</li><li>Iwase, Y., Maitani, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation+and+in+vivo+evaluation+of+liposomal+everolimus+for+lung+carcinoma+and+thyroid+carcinoma.">Preparation and in vivo evaluation of liposomal everolimus for lung carcinoma and thyroid carcinoma.</a> Biol Pharm Bull. 35, 975-979 (2012).</li><li>Mishra, G. P., Doddapaneni, B. S., Nguyen, D., Alani, A. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antiangiogenic+effect+of+docetaxel+and+everolimus+as+individual+and+dual-drug-loaded+micellar+nanocarriers.">Antiangiogenic effect of docetaxel and everolimus as individual and dual-drug-loaded micellar nanocarriers.</a> Pharm Res. 31, 660-669 (2014).</li><li>Xu, W., Ling, P., Zhang, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Polymeric+micelles,+a+promising+drug+delivery+system+to+enhance+bioavailability+of+poorly+water-soluble+drugs.">Polymeric micelles, a promising drug delivery system to enhance bioavailability of poorly water-soluble drugs.</a> J Drug Deliv. 2013, 340315 (2013).</li><li>Lavasanifar, A., Samuel, J., Kwon, G. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Poly(ethylene+oxide)-block-poly(L-amino+acid)+micelles+for+drug+delivery.">Poly(ethylene oxide)-block-poly(L-amino acid) micelles for drug delivery.</a> Adv Drug Deliv Rev. 54, 169-190 (2002).</li></ol>

The authors have nothing to disclose.

The use of polymeric micelles for drug delivery continues to expand due to their versatility and ability to deliver hydrophobic drugs for various disease states. Therefore, the techniques needed to prepare and characterize these formulations prior to use in cell culture or animals is a critical first step to determine the best pairing between the drug and the polymer. PEG-b-PLA are excellent amphiphilic block copolymers for drug delivery purposes. However, the block length of the hydrophilic and hydrophobic s...

קופולימרים לחסום amphiphilic עם מבנה חוזר מורכבים מתחומים הידרופילי והידרופובי יכולים באופן ספונטני עצמיים להרכיב-כדי ליצור שלושה מכלולי macromolecular ממדיים הידוע במיצלות פולימרים. יש מבנים אלה ליבה הידרופובי פנימית מוקפות פגז הידרופילי. יש הליבה הידרופובי היכולת לשלב תרופות הידרופובי או על ידי לכידה פיזית באמצעות אינטראקציות הידרופוביות או על ידי נטיה כימית לשדרת הפולימר. 1 יתרונות רבים קיימים לשימוש בקופולימרים לחסום אלה כדי ליצור מיצלות עבור משלוח סמים. אלה כוללים שילוב של תרופות מסיסים היטב, שיפור הפרמקוקינטיקה של התרופות משולבות, והתאמה ביולוגיות ו / או פריקות ביולוגית של הפולימרים גורם להם חלופי בטוח solubilizers הקונבנציונלי. 2 יתרון נוסף של שימוש במיצלות פולימרים הוא גודל החלקיקים קולואידים, בין 15 150 3 ננומטר, מה שהופך אותם אטרקטיביים לרשות פלסטיניתמשלוח renteral. לכן, ב -20 השנים האחרונות מיצלות פולימרים צמחה כמערכות אספקת סמים ברות-קיימא עבור תרופות מסיסים במים בצורה גרועה במיוחד לטיפול בסרטן. 3,4 כרגע יש חמישה ניסוחים micellar פולימרים לטיפול בסרטן בניסויים קליניים. 4 ארבעה של מיצלות בניסויים הקליניים קופולימרים diblock מבוססות PEG בעוד האחרון הוא קופולימר triblock מכיל polyethyleneoxide. הגודל של מיצלות אלה נע בין 20 ננומטר ל -85 ננומטר. היתרון של שימוש בפולימרים מבוססים PEG הוא ההתאמה הביולוגית שלהם ובהתאם לבלוק השני יכול להיות גם מתכלה. לאחרונה מערכות אספקת הסמים חדשות המבוסס על חומצת -polylactic בלוק polyethyleneglycol- (PEG- ב -PLA) מיצלות פולימרים פותחו עבור המשלוח המקביל של תרופות נגד סרטן מרובים. מיצלות PEG- B- PLA שניהם ביולוגית ומתכלה. רב-סמים אלה מיצלות העמוסה הראו כעיכוב ynergistic של דגמי סוגי הסרטן שונים במבחנת in vivo 2,5,6 ובכושר לפרדיגמה הנוכחית של ניצול תרופות מרובות בכימותרפיה כדי למנוע התנגדות והוריד רעילות. לכן, יש עניין רב בהכנה ואפיון מערכות אספקת הסמים micellar אלה לשימוש בסרטן ומצבי מחלה אחרים. בעבודה שלהלן שהתווינו תהליך צעד-אחר-צעד שיכולה להיות מוכן מיצלות כזה ומאופיין לפני הערכתם במצבי מחלה של עניין. לצורך העבודה זו שני סוכנים אנטי-סרטניים גרוע מסיסים, docetaxel (DTX) וeverolimus (EVR) נבחרו. DTX וEVR שניהם תרכובות מסיסים במים בצורה גרועה עם solubilities מים פנימי וב1.9 9.6 מיקרוגרם / מיליליטר בהתאמה. 7,8 שני פולימרים -PLA ב PEG- עם משקולות מולקולריות שונות ששימשו בפרוטוקול זה כאבני בניין לפולימרים שגובשו מיצלות,פולימרים אלו PEG 2000 - ב -PLA 1,800 (3,800 Da) וPEG 4000 - ב -PLA 2,200 (6,200 Da). מיצלות -PLA ב PEG- לכן יכולה לספק פלטפורמה ייחודית כnanocarrier לDTX וEVR בנפרד ובשילוב. ריאגנטים / חומרים והציוד הדרושים להכנה ולאפיין מיצלות אלה מוצגות בטבלה 1.

<table><tbody><tr><td>PEG2000-b-PLA1800</td><td>Advanced Polymer Materials, Inc</td><td>6-01- PLA/2000</td><td>PLA MW can be specified on ordering</td></tr><tr><td>PEG4000-b-PLA2200</td><td>Advanced Polymer Materials, Inc</td><td>6-01- PLA/4000</td><td>PLA MW can be specified on ordering</td></tr><tr><td>Docetaxel</td><td>LC Laboratories</td><td>D-1000</td><td>100 mg</td></tr><tr><td>Everolimus</td><td>LC Laboratories</td><td>E-4040</td><td>100 mg</td></tr><tr><td>Acetonitrile</td><td>EMD/VWR</td><td>EM-AX0145-1</td><td>HPLC grade; 4 L</td></tr><tr><td>Round bottom flask&amp;nbsp;</td><td>Glassco/VWR</td><td>89426-496</td><td>5 ml</td></tr><tr><td>RV 10 Control Rotary Evaporators</td><td>IKA Works</td><td>8025001</td><td>Rotoevaporator</td></tr><tr><td>Shimadzhu HPLC with DAD detector</td><td>Shimadzhu</td><td></td><td>RP-HPLC</td></tr><tr><td>Slide-a-lyzer dialysis casette MWCO 7000</td><td>Thermo Scientific, Inc</td><td>66370</td><td>3 ml</td></tr><tr><td>Phosphate buffer pH 7.4, 200 mM</td><td>VWR</td><td>100190-870</td><td>500 ml</td></tr><tr><td>Malvern NanoZS</td><td>Malvern Instruments, UK</td><td></td><td>DLS</td></tr><tr><td>Nylon filter</td><td>Acrodisc/VWR</td><td>28143-242</td><td>13 mm; 0.2&amp;micro;M</td></tr><tr><td>Phosphoric acid, NF</td><td>Spectrum Chemical/VWR</td><td>700000-626</td><td>100 ml</td></tr><tr><td>GraphPad Prism</td><td>&lt;a href=&quot;http://www.graphpad.com/&quot;&gt;www.graphpad.com&lt;/a&gt;</td><td></td><td>Analysis software</td></tr><tr><td>Zorbax SB-C8 Rapid Resolution cartridge&amp;nbsp;</td><td>Agilent Technologies</td><td>866953-906</td><td>4.6 &amp;times;75 mm, 3.5 &amp;#956;m</td></tr></tbody></table>

preparation and characterization of individual and multi-drug loaded physically entrapped polymeric micelles

קופולימרים לחסום amphiphilic כמו חומצת -polylactic בלוק polyethyleneglycol- (PEG- B- PLA) יכולים עצמי להרכיב למיצלות מעל ריכוז micellar הקריטי שלהם ויוצרת ליבות הידרופובי מוקפות פגזים הידרופילי בסביבות מימיות. הליבה של מיצלות אלה יכול להיות מנוצל כדי לטעון תרופות הידרופובי, גרועה מסיסה במים כמו docetaxel (DTX) וeverolimus (EVR). אפיון שיטתי של יכולות מבנה micelle וטעינת תרופה חשוב לפני במבחנה ובמחקרי vivo יכול להתנהל. המטרה של הפרוטוקול המתואר במסמך זה היא לספק את הצעדים הדרושים כדי להשיג אפיון מוצרי micellar סטנדרטיים. יש לי DTX וEVR solubilities הפנימי של 1.9 ו -9.6 מיקרוגרם / מיליליטר בהתאמה ניתן להשיג הכנת מיצלות אלה באמצעות יציקת ממס המגדילה את המסיסות המימית של DTX וEVR ל1.86 ו1.85 מ&quot;ג / מיליליטר, בהתאמה. יציבות בסמים במיצלות evaluated בטמפרטורת חדר במשך 48 שעות עולה כי 97% או יותר מהתרופות נשמרים בפתרון. גודל micelle הוערך באמצעות פיזור אור דינאמי והצביע על כך שהגודל של מיצלות אלה היה מתחת ל -50 ננומטר ותלויים במשקל המולקולרי של הפולימר. שחרור תרופה ממיצלות הוערך באמצעות דיאליזה בתנאי כיור ב- pH 7.4 ב 37 מעלות צלזיוס במשך 48 שעות. תוצאות ראויים עקומה מצביעות על כך ששחרור תרופה מונע על ידי תהליך הזמנה ראשון מצביע על כך שהוא מונע דיפוזיה.

DTX בודד או מיצלות EVR וDTX וEVR DDM בPEG- ב -PLA מיצלות מנוסחות בהצלחה בשני PEG 4000 - ב -PLA 2,200 או PEG 2000 - ב -PLA 1,800 (איור 1). DTX, EVR, וDDM הראו יציבות דומה בPEG 4000 - ב -PLA 2,200 או PEG 2000 - ב...

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Preparation and Characterization of Individual and Multi-drug Loaded Physically Entrapped Polymeric Micelles

The goal of this protocol is to describe the preparation and characterization of physically entrapped, poorly water soluble drugs in micellar drug delivery systems composed of amphiphilic block copolymers.

הכנה ואפיון של פרט ורב-סמים נטענים בפח פיזי Micelles פולימרית

Amphiphilic block copolymers like polyethyleneglycol-block-polylactic acid (PEG-b-PLA) can self-assemble into micelles above their critical micellar ...

preparation-characterization-individual-multi-drug-loaded-physically

Universidad San Sebastian

Research

JoVE Journal

Chemistry

11.2K Views.  Pacific University. The goal of this protocol is to describe the preparation and characterization of physically entrapped, poorly water soluble drugs in micellar drug delivery systems composed of amphiphilic block copolymers.

Video: Preparation and Characterization of Individual and Multi-drug Loaded Physically Entrapped Polymeric Micelles

A Facile and Efficient Approach for the Production of Reversible Disulfide Cross-linked Micelles

Nanomedicine is an emerging form of therapy that harnesses the unique properties of particles that are nanometers in scale for biomedical application. Improving drug delivery to maximize therapeutic outcomes and to reduce drug-associated side effects are some of the cornerstones of present-day nanomedicine. Nanoparticles in particular have found a wide application in cancer treatment. Nanoparticles that offer a high degree of flexibility in design, application, and production based on the tumor microenvironment are projected to be more effective with rapid translation into clinical practice. The polymeric micellar nano-carrier is a popular choice for drug delivery applications.
In this article, we describe a simple and effective protocol for synthesizing drug-loaded, disulfide cross-linked micelles based on the self-assembly of a well-defined amphiphilic linear-dendritic copolymer (telodendrimer, TD). TD is composed of polyethylene glycol (PEG) as the hydrophilic segment and a thiolated cholic acid cluster as the core-forming hydrophobic moiety attached stepwise to an amine-terminated PEG using solution-based peptide chemistry. Chemotherapy drugs, such as paclitaxel (PTX), can be loaded using a standard solvent evaporation method. The O2-mediated oxidation was previously utilized to form intra-micellar disulfide cross-links from free thiol groups on the TDs. However, the reaction was slow and not feasible for large-scale production. Recently, an H2O2-mediated oxidation method was explored as a more feasible and efficient approach, and it was 96 times faster than the previously reported method. Using this approach, 50 g of PTX-loaded, disulfide cross-linked nanoparticles have been successfully produced with narrow particle size distribution and high drug loading efficiency. The stability of the resulting micelle solution is analyzed using disrupting conditions such as co-incubation with a detergent, sodium dodecyl sulfate, with or without a reducing agent. The drug-loaded, disulfide cross-linked micelles demonstrated less hemolytic activity when compared to their non-cross-linked counterparts.

To deliver cancer drugs to tumor sites with high specificity and reduced side effects, new methods based on nanoparticles are required. Here, we describe disulfide cross-linked micelles that can be easily prepared by hydrogen peroxide-mediated oxidation and are able to dissociate efficiently under a reducing tumor environment to release payloads.

גישה קלילה ויעילה לייצור ההפיך דיסולפיד צולב Micelles

Nanomedicine is an emerging form of therapy that harnesses the unique properties of particles that are nanometers in scale for biomedical application. ...

Biochemistry

Formulation of Diblock Polymeric Nanoparticles through Nanoprecipitation Technique

Nanotechnology is a relatively new branch of science that involves harnessing the unique properties of particles that are nanometers in scale (nanoparticles). Nanoparticles can be engineered in a precise fashion where their size, composition and surface chemistry can be carefully controlled. This enables unprecedented freedom to modify some of the fundamental properties of their cargo, such as solubility, diffusivity, biodistribution, release characteristics and immunogenicity. Since their inception, nanoparticles have been utilized in many areas of science and medicine, including drug delivery, imaging, and cell biology1-4. However, it has not been fully utilized outside of "nanotechnology laboratories" due to perceived technical barrier. In this article, we describe a simple method to synthesize a polymer based nanoparticle platform that has a wide range of potential applications. 
The first step is to synthesize a diblock co-polymer that has both a hydrophobic domain and hydrophilic domain. Using PLGA and PEG as model polymers, we described a conjugation reaction using EDC/NHS chemistry5 (Fig 1). We also discuss the polymer purification process. The synthesized diblock co-polymer can self-assemble into nanoparticles in the nanoprecipitation process through hydrophobic-hydrophilic interactions.
The described polymer nanoparticle is very versatile. The hydrophobic core of the nanoparticle can be utilized to carry poorly soluble drugs for drug delivery experiments6. Furthermore, the nanoparticles can overcome the problem of toxic solvents for poorly soluble molecular biology reagents, such as wortmannin, which requires a solvent like DMSO. However, DMSO can be toxic to cells and interfere with the experiment. These poorly soluble drugs and reagents can be effectively delivered using polymer nanoparticles with minimal toxicity. Polymer nanoparticles can also be loaded with fluorescent dye and utilized for intracellular trafficking studies. Lastly, these polymer nanoparticles can be conjugated to targeting ligands through surface PEG. Such targeted nanoparticles can be utilized to label specific epitopes on or in cells7-10.

This article describes a nanoprecipitation method to synthesize polymer-based nanoparticles using diblock co-polymers. We will discuss the synthesis of diblock co-polymers, the nanoprecipitation technique, and potential applications.

גיבוש חלקיקים פולימריים Diblock באמצעות טכניקה Nanoprecipitation

Nanotechnology is a relatively new branch of science that involves harnessing the unique properties of particles that are nanometers in scale ...

Bioengineering

Preparation and Characterization of SDF-1&#945;-Chitosan-Dextran Sulfate Nanoparticles

Chitosan (CS) and dextran sulfate (DS) are charged polysaccharides (glycans), which form polyelectrolyte complex-based nanoparticles when mixed under appropriate conditions. The glycan nanoparticles are useful carriers for protein factors, which facilitate the in vivo delivery of the proteins and sustain their retention in the targeted tissue. The glycan polyelectrolyte complexes are also ideal for protein delivery, as the incorporation is carried out in aqueous solution, which reduces the likelihood of inactivation of the proteins. Proteins with a heparin-binding site adhere to dextran sulfate readily, and are, in turn, stabilized by the binding. These particles are also less inflammatory and toxic when delivered in vivo. In the protocol described below, SDF-1&#945; (Stromal cell-derived factor-1&#945;), a stem cell homing factor, is first mixed and incubated with dextran sulfate. Chitosan is added to the mixture to form polyelectrolyte complexes, followed by zinc sulfate to stabilize the complexes with zinc bridges. The resultant SDF-1&#945;-DS-CS particles are measured for size (diameter) and surface charge (zeta potential). The amount of the incorporated SDF-1&#945; is determined, followed by measurements of its in vitro release rate and its chemotactic activity in a particle-bound form.

The objective of this protocol is to incorporate SDF-1&#945;, a stem cell homing factor, into dextran sulfate-chitosan nanoparticles. The resultant particles are measured for their size and zeta potential, as well as the content, activity, and in vitro release rate of SDF-1&#945; from the nanoparticles.

הכנה ואפיון של SDF-1α-Chitosan-Dextran סולפט חלקיקים

Chitosan (CS) and dextran sulfate (DS) are charged polysaccharides (glycans), which form polyelectrolyte complex-based nanoparticles when mixed under ...

Facile Preparation of Internally Self-assembled Lipid Particles Stabilized by Carbon Nanotubes

We present a facile method to prepare nanostructured lipid particles stabilized by carbon nanotubes (CNTs). Single-walled (pristine) and multi-walled (functionalized) CNTs are used as stabilizers to produce Pickering type oil-in-water (O/W) emulsions. Lipids namely, Dimodan U and Phytantriol are used as emulsifiers, which in excess water self-assemble into the bicontinuous cubic Pn3m phase. This highly viscous phase is fragmented into smaller particles using a probe ultrasonicator in presence of conventional surfactant stabilizers or CNTs as done here. Initially, the CNTs (powder form) are dispersed in water followed by further ultrasonication with the molten lipid to form the final emulsion. During this process the CNTs get coated with lipid molecules, which in turn are presumed to surround the lipid droplets to form a particulate emulsion that is stable for months. The average size of CNT-stabilized nanostructured lipid particles is in the submicron range, which compares well with the particles stabilized using conventional surfactants. Small angle X-ray scattering data confirms the retention of the original Pn3m cubic phase in the CNT-stabilized lipid dispersions as compared to the pure lipid phase (bulk state). Blue shift and lowering of the intensities in characteristic G and G&#39; bands of CNTs observed in Raman spectroscopy characterize the interaction between CNT surface and lipid molecules. These results suggest that the interactions between the CNTs and lipids are responsible for their mutual stabilization in aqueous solutions. As the concentrations of CNTs employed for stabilization are very low and lipid molecules are able to functionalize the CNTs, the toxicity of CNTs is expected to be insignificant while their biocompatibility is greatly enhanced. Hence the present approach finds a great potential in various biomedical applications, for instance, for developing hybrid nanocarrier systems for the delivery of multiple functional molecules as in combination therapy or polytherapy.

We report on a smart application of carbon nanotubes for kinetic stabilization of lipid particles that contain self-assembled nanostructures in their cores. The preparation of lipid particles requires rather low concentrations of carbon nanotubes permitting their use in biomedical applications such as drug delivery.

הכנה קלילה של פנימית עצמית התאספו חלקיקים ליפידים מיוצב על ידי פחמן

We present a facile method to prepare nanostructured lipid particles stabilized by carbon nanotubes (CNTs). Single-walled (pristine) and multi-walled ...

Anionic Polymerization of an Amphiphilic Copolymer for Preparation of Block Copolymer Micelles Stabilized by &#960;-&#960; Stacking Interactions

In this study, an amphiphilic copolymer that includes a core-forming block with phenyl groups was synthesized by living anionic polymerization of phenyl glycidyl ether (PheGE) on methoxy-polyethylene glycol (mPEG-b-PPheGE). Characterization of the copolymer revealed a narrow molecular distribution (PDI &#60; 1.03) and confirmed the degree of polymerization of mPEG122-b-(PheGE)15. The critical micelle concentration of the copolymer was evaluated using an established fluorescence method with the aggregation behavior evaluated by dynamic light scattering and transmission electronic microscopy. The potential of the copolymer for use in drug delivery applications was evaluated in a preliminary manner including in vitro biocompatibility, loading and release of the hydrophobic anti-cancer drug doxorubicin (DOX). A stable micelle formulation of DOX was prepared with drug loading levels up to 14% (wt%), drug loading efficiencies &#62; 60% (w/w) and sustained release of drug over 4 days under physiologically relevant conditions (acidic and neutral pH, presence of albumin). The high drug loading level and sustained release is attributed to stabilizing &#960;-&#960; interactions between DOX and the core-forming block of the micelles.

The key steps of living anionic polymerization of phenyl glycidyl ether (PheGE) on methoxy-polyethylene glycol (mPEG-b-PPheGE) are described. The resulting block copolymer micelles (BCMs) were loaded with doxorubicin 14% (wt%) and sustained release of drug over 4 days under physiologically relevant conditions was obtained.

Anionic פילמור של קופולימר amphiphilic עבור הכנת Micelles קופולימר בלוק מיוצב על ידי אינטראקציות הנחת π-π

In this study, an amphiphilic copolymer that includes a core-forming block with phenyl groups was synthesized by living anionic polymerization of phenyl ...

Intra-lymph Node Injection of Biodegradable Polymer Particles

Generation of adaptive immune response relies on efficient drainage or trafficking of antigen to lymph nodes for processing and presentation of these foreign molecules to T and B lymphocytes. Lymph nodes have thus become critical targets for new vaccines and immunotherapies. A recent strategy for targeting these tissues is direct lymph node injection of soluble vaccine components, and clinical trials involving this technique have been promising. Several biomaterial strategies have also been investigated to improve lymph node targeting, for example, tuning particle size for optimal drainage of biomaterial vaccine particles. In this paper we present a new method that combines direct lymph node injection with biodegradable polymer particles that can be laden with antigen, adjuvant, or other vaccine components. In this method polymeric microparticles or nanoparticles are synthesized by a modified double emulsion protocol incorporating lipid stabilizers. Particle properties (e.g.&#160;size, cargo loading) are confirmed by laser diffraction and fluorescent microscopy, respectively. Mouse lymph nodes are then identified by peripheral injection of a nontoxic tracer dye that allows visualization of the target injection site and subsequent deposition of polymer particles in lymph nodes. This technique allows direct control over the doses and combinations of biomaterials and vaccine components delivered to lymph nodes and could be harnessed in the development of new biomaterial-based vaccines.

Lymph nodes are the immunological tissues that orchestrate immune response and are a critical target for vaccines. Biomaterials have been employed to better target lymph nodes and to control delivery of antigens or adjuvants. This paper describes a technique combining these ideas to inject biocompatible polymer particles into lymph nodes.

הזרקת בלוטות תוך-לימפתיות של חלקיקי פולימר מתכלים

Generation of adaptive immune response relies on efficient drainage or trafficking of antigen to lymph nodes for processing and presentation of these ...

Assembly and Characterization of Polyelectrolyte Complex Micelles

Polyelectrolyte complex micelles (PCMs), core-shell nanoparticles formed by self-assembly of charged polymers in aqueous solution, provide a powerful platform for exploring the physics of polyelectrolyte interactions and also offer a promising solution to the pressing problem of delivering therapeutic oligonucleotides in vivo. Developing predictive structure-property relationships for PCMs has proven difficult, in part due to the presence of strong kinetic traps during nanoparticle self-assembly. This article discusses criteria for choosing polymers for PCM construction and provides protocols based on salt annealing that enable assembly of repeatable, low-polydispersity nanoparticles. We also discuss PCM characterization using light scattering, small-angle X-ray scattering, and electron microscopy.

We provide protocols and representative data for designing, assembling, and characterizing polyelectrolyte complex micelles, core-shell nanoparticles formed by polyelectrolytes and hydrophilic charged-uncharged block copolymers.

הרכבה ואפיון של מטלות מורכבות פוליאלקטרוליט

Polyelectrolyte complex micelles (PCMs), core-shell nanoparticles formed by self-assembly of charged polymers in aqueous solution, provide a powerful ...

Preparation and Characterization of Lipophilic Doxorubicin Pro-drug Micelles

Micelles have been successfully used for the delivery of anticancer drugs. Amphiphilic polymers form core-shell structured micelles in an aqueous environment through self-assembly. The hydrophobic core of micelles functions as a drug reservoir and encapsulates hydrophobic drugs. The hydrophilic shell prevents the aggregation of micelles and also prolongs their systemic circulation in vivo. In this protocol, we describe a method to synthesize a doxorubicin lipophilic pro-drug, doxorubicin-palmitic acid (DOX-PA), which will enhance drug loading into micelles. A pH-sensitive hydrazone linker was used to conjugate doxorubicin with the lipid, which facilitates the release of free doxorubicin inside cancer cells. Synthesized DOX-PA was purified with a silica gel column using dichloromethane/methanol as the eluent. Purified DOX-PA was analyzed with thin layer chromatography (TLC) and 1H-Nuclear Magnetic Resonance Spectroscopy (1H-NMR). A film dispersion method was used to prepare DOX-PA loaded DSPE-PEG micelles. In addition, several methods for characterizing micelle formulations are described, including determination of DOX-PA concentration and encapsulation efficiency, measurement of particle size and distribution, and assessment of in vitro anticancer activities. This protocol provides useful information regarding the preparation and characterization of drug-loaded micelles and thus will facilitate the research and development of novel micelle-based cancer nanomedicines.

A protocol for the preparation and characterization of lipophilic doxorubicin pro-drug loaded 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)-2000] (DSPE-PEG) micelles is described.

הכנה ואפיון של דוקסורוביצין lipophilic Micelles פרו-סמים

Micelles have been successfully used for the delivery of anticancer drugs. Amphiphilic polymers form core-shell structured micelles in an aqueous ...

Phase Change Dimethyldioctadecylammonium-Shelled Microdroplets as a Promising Drug Delivery System: Results on 3D Spheroids of Mammalian Tumor Cells

Significant improvement of phase-change perfluorocarbon microdroplets (MDs) in the vast theranostic scenario passes through the optimization of the MDs composition with respect to synthesis efficiency, stability, and drug delivery capability. To this aim, decafluoropentane (DFP) MDs stabilized by a shell of dimethyldioctadecylammonium bromide (DDAB) cationic surfactant were designed. A high concentration of DDAB-MDs was readily obtained within a few seconds by pulsed high-power insonation, resulting in low polydisperse 1 &#181;m size droplets. Highly positive &#950;-potential, together with a long, saturated hydrocarbon chains of the DDAB shell, are key factors to stabilize the droplet and the drug cargo therein. The high affinity of the DDAB shell with cell plasma membrane allows for localized chemotherapeutics delivery by increasing the drug concentration at the tumor cell interface and boosting the uptake. This would turn DDAB-MDs into a relevant drug delivery tool exhibiting high antitumor activity at very low drug doses.
In this work, the efficacy of such an approach is shown to dramatically improve the effect of doxorubicin against 3D spheroids of mammalian tumor cells, MDA-MB-231. The use of&#160;three-dimensional (3D) cell cultures developed in the form of multicellular tumor spheroids (i.e., densely packed cells in a spherical shape) has numerous advantages compared to 2D cell cultures: in addition to have the potential to bridge the gap between conventional in vitro studies and animal testing, it will improve the ability to perform more predictive in vitro screening assays for preclinical drug development or evaluate the potential of off-label drugs and new co-targeting strategies.

Decafluoropentane microdroplets developed with a shell of dimethyldioctadecylammonium bromide exhibited an exceptional colloidal stability and an actractive biointerface. DDAB-MDs proved to be efficient drug reservoirs characterized by a high affinity to plasma membranes together with enhanced uptake and antitumor activity of Doxorubicin against human triple-negative breast cancer (MDA-MB-231) 3D model.

Significant improvement of phase-change perfluorocarbon microdroplets (MDs) in the vast theranostic scenario passes through the optimization of the MDs ...

Preparation and Characterization of Nanoliposomes for the Entrapment of Bioactive Hydrophilic Globular Proteins

Liposome nanocapsules have been applied for many purposes in the pharmaceutical, cosmetic, and food industries. Attributes of liposomes include their biocompatibility, biodegradability, non-immunogenicity, non-toxicity, and ability to entrap both hydrophilic and hydrophobic compounds. The classical hydration of thin lipid films in an organic solvent is applied herein as a technique to encapsulate tarin, a plant lectin, in nanoliposomes. Nanoliposome size, stability, entrapment efficiency, and morphological characterization are described in detail. The nanoliposomes are prepared using 1,2-dioleoyl-sn-glycerol-3-phosphoethanolamine (DOPE), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)-2000] (ammonium salt; DSPE-MPEG 2000), and cholesterylhemisuccinate (CHEMS) as the main constituents. Lipids are first dissolved in chloroform to obtain a thin lipid film that is subsequently rehydrated in ammonium sulfate solution containing the protein to be entrapped and incubated overnight. Then, sonication and extrusion techniques are applied to generate nanosized unilamellar vesicles. The size and polydispersity index of the nanovesicles are determined by dynamic light scattering, while nanovesicle morphology is assessed by scanning electron microscopy. Entrapment efficiency is determined by the ratio of the amount of unencapsulated protein to original amount of initially loaded protein. Homogeneous liposomes are obtained with an average size of 155 nm and polydispersity index value of 0.168. A high entrapment efficiency of 83% is achieved.

This study describes classical hydration using the thin lipid film method for nanoliposome preparation followed by nanoparticle characterization. A 47 kDa-hydrophilic and globular protein, tarin, is successfully encapsulated as a strategy to improve stability, avoid fast clearance, and promote controlled release. The method can be adapted to hydrophobic molecules encapsulation.

הכנה ואפיון ננו-פוטזומים לצורך מלכודת הידרופיזיאליות של חלבונים ביולוגיים

Liposome nanocapsules have been applied for many purposes in the pharmaceutical, cosmetic, and food industries. Attributes of liposomes include their ...