This work was supported by the following grants: NIH-SC3 grant, NSF-PREM grant, Hampton University Faculty Research Grant. We would like to thank Mrs. Michele A. Cochran at Virginia Institute of Marine Science (VIMS) for the use of the particle size analyzer. We would also like to thank Mrs. Corinne R. Ramaley for reviewing the manuscript.

DOX-PA 1. Sentezi <ol><li> Doksorubisinin 390 mg ve palmitik asit hidrazid 243 mg ağırlığında ve yuvarlak dipli bir şişeye aktarın. </li><li> cam şırınga ile şişeye susuz metanol 150 ml ekleyin. bir pipet ile trifloroasetik asit (TFA) 39 ul ekle. Manyetik bir karıştırıcı kullanarak, karanlıkta, oda sıcaklığında 18 saat boyunca reaksiyon karışımının karıştırın. NOT: Reaksiyon malzemelerin miktarları kadar ölçeklendirilebilir veya aşağı DOX-PA farklı miktarlarda elde etmek için. reaktiflerin oranı, aynı oranlarda muhafaza edilmelidir. 78 mg 1.170 mg aralığında DOX miktarlar kullanılarak reaksiyonlar düzenli kimya laboratuvar gerçekleştirilebilir. </li><li> Bir silika jel kolonu kullanılarak DOX-PA ile saflaştırılması. 17. <ol><li> Bir dönen buharlaştıncı ile reaksiyon karışımında çözücüyü çıkarın. Karışımın hacmi yaklaşık 20 ml azaltılmış sonra, silika jel, 3 g ekleyin. kuru tozlar elde etmek için döner buharlaştırma devam edin ve t sağlamak içinO silika jel üzerine ürünlerin adsorplanmadan. Kuru tozlar oluştuktan sonra ilave bir 30 dakika boyunca vakum altında örnek tutun. </li><li> bir çözücü olarak diklorometan kullanılarak bir sütun halinde, silika jel 50 g paketleyin. Dikkatle kolonuna adsorbe ürün silika jel içeren bir örnek ilave edin. </li><li> Yavaş yavaş ve böylece çözücünün polaritesi (Tablo 1), artan metanol yüzdesi artırırken, bir diklorometan ve metanol karışımı ile sütun yıkayın. </li><li> Test tüpleri içinde eluent fraksiyonlar (25 mi / tüp) toplayın ve ince tabaka kromatografisi (TLC) ile ilerlemesini izlemek. </li><li> Saf DOX-PA içeren tüm fraksiyonlar birleştirilir ve kuru toz elde edilinceye kadar, bir döner evaporatör kullanılarak solvent çıkartılmıştır. Bundan başka, bir vakum O / N ve ürünle kurutun. </li></ol></li><li> TLC ile DOX-PA analizi. <ol><li> TLC plakası bir 4 cm x 8 cm uzunluğunda kesilir. TLC tespit kılcal damarların ile plakanın alt spot örnek çözümler 0.5 cm buluştu kullanarakçözücü olarak HANOL. </li><li> diklorometan ve metanol karışımı içeren bir geliştirme odasına TLC plakasını (3/1, hac / hac). Çözücünün derinliği 0.5 cm sadece az olmalıdır. </li><li> Çözücü ön plaka üst ulaştığında gelişmekte odasından plakasını sökün. Bir kalemle çözücü ön yerini işaretlemek ve plaka kurumasını bekleyin. örnekleri görselleştirmek için doymuş iyodin buharı ihtiva eden bir boyama odasına TLC plakasını. </li></ol></li><li> 1H-Nükleer Manyetik Rezonans Spektroskopisi (1 &#39;H-NMR) ile DOX-PA analizi. 18 <ol><li> metil sülfoksit-d6 (DMSO) içinde 1 ml DOX-PS 15 mg çözülür ve bir NMR tüpü içine örnek aktarmak. </li><li> NMR aracın cazibe merkezi haline NMR tüpü yerleştirin. Bir çözücü olarak DMSO seçilmesi, proton spektrumunun ölçün. mıknatıstan NMR tüpü çıkarın. NMR sonucu 18 analiz edin. </li></ol></li></ol> 2. Film dağıtım metodu ile DOX-PA miseller hazırlanması <ol><li> DSPE-PEG (40 mg) ve DOX-PA (4 mg), 10 ml bir cam şişe içinde, 2 ml metanol ile çözülür. </li><li> şişede ince bir film oluşana kadar bir döner buharlaştırıcı kullanılarak vakum altında organik çözücüyü çıkarın. Alternatif olarak, (örneğin, argon veya azot gazı), bir film oluşturmak ve daha fazla kalan solventin çıkması için bir vakum desikatörde flakon tutmak için atıl gaz altında, organik çözücüler buharlaştırılmış: NOT. </li><li> Aktarım cam şişeye Dulbecco fosfat tamponlu tuzlu su (pH 7.4, DPBS) içinde 2 mi. </li><li> miseller oluşturmak için oda sıcaklığında 3 dakika boyunca ultrasonik bir banyoda şişe yerleştirin. NOT: Ultrasonik güç ultrasonik banyolarda farklı modeller arasında değişir. ince polimer / ilaç filmi dağıtmak için yeterli ultrasonik güç üretebilir bir birimi seçin. Bu protokolde kullanılan ultrasonik banyosunun çıkış gücü 110 W olan </li><li> Kısa süreli depolama için 4 ° C&#39;de miseller tutmak ve -20 ° C de, uzunuzun dönem depolama. Not: Alternatif olarak, misellerin dondurularak kurutulmuş ve kullanımdan önce su ile yeniden oluşturulabilir. Genellikle, herhangi bir dondurarak saklama koruyucusu ve liyoprotektan, bu formülasyon için gereklidir. </li></ol> DOX-PA misellerin 3. karakterizasyonu <ol><li> Miseller ve ilaç kapsülleme verimliliği DOX-PA konsantrasyonunun belirlenmesi <ol><li> 1 ug / ml, 5 ug / ml, 20 ug / ml, 50 ug / ml ve 100 ug / ml: DOX-PA beş farklı konsantrasyonlarda DOX-PA çözeltiler hazırlanması DMSO içinde bir önceki adımda sentezlenmiştir çözülür. 490 nm&#39;de bir UV-VIS spektrometresi ile DOX-PA çözeltiler emilimini belirlemek. DOX-PA madde konsantrasyonları ve 490 nm&#39;de bunların karşılık gelen emme göre bir standart eğri oluşturur. </li><li> DMSO, 500 ul ilaç yüklü misel 25 ul seyreltilir. Bir UV-VIS spektrometresi ile 490 nm&#39;de emilim ölçülür. 3.1.1 oluşturulan standart eğri ile ilaç konsantrasyonlarını hesaplayın. </li><li> Aşağıdaki denklem kullanılarak Kapsülleme verimliliği hesaplayın: İlaç Kapsülleme verimliliği (%) = (miseller ilaç miktarı) / (ilave ilaç miktarı) x% 100 </li></ol></li><li> Dinamik ışık dağılımı ile parçacık boyutunun Karakterizasyonu (DLS) <ol><li> 1 mg / ml&#39;lik bir son DSPE-PEG konsantrasyonuna DPBS (pH 7.4) ile miseller seyreltilir. Z-ortalama boyutu ve çok yönlü dağılma indeksi (PDI) elde etmek için bir parçacık boyutu analiz cihazı ile, 2 ml lik bir numuneyi analiz edin. </li></ol></li><li> In vitro anti-kanser etkinliğinin değerlendirilmesi NOT: Uygun steril tekniği kullanın ve bir biyogüvenlik kabini içinde çalışırlar. <ol><li> Bir hücre kültür şişesine (örn, T25) ihtiva eden, DU-145, insan prostat kanseri hücreleri, hücre kültürü ortamının çıkarın ve DPBS, 2 ml (pH 7.4) ile hücrelerin yıkayın. </li><li> Aspire DPBS, tripsin solüsyonu (% 0.25) 1 ml ekleyin ve hücreleri ayırmak için 37 ° C&#39;de 2 dakika süreyle inkübe edin. </li><li> 10 m eklehücre kültürü aracı maddesi (RPMI 1640 +% 10 cenin sığır serumu +% 1 antibiyotik antimikotik), hücrelerin en şişeden çözülmüş olduğunda. 15 ml santrifüj tüpüne ve santrifüj 5 dakika boyunca 1000 x g&#39;de hücreleri hücreleri aktarın. </li><li> Hücre kültür ortamı, 5 ml hücre pelet yeniden askıya ve bir hemositometrede hücre adedini saymak için bir örnek çıkarın. 50,000 hücre / ml&#39;lik bir yoğunluğa kadar hücre kültürü ortamı ile hücreleri seyreltilir. Bir 96-yuvalı hücre kültürü plakasının seyreltilmiş hücre süspansiyonu ekleyin (100 ul / oyuk). Bir hücre kültür inkübatörü içinde inkübe hücreleri (37 ° C,% 5 CO2) 18 saat hücre tutunmasını sağlar. </li><li> sırasıyla, 0.1 uM ve 0.5 uM, 2 uM, 5 uM ve 10 uM nihai ilaç konsantrasyonlarını elde etmek için hücre kültürü ortamı ile DOX dimetil sülfoksit (DMSO) çözeltisi ve DOX-PA DMSO çözeltisi ile seyreltilir. % 0.5 her şeyden örneklerde nihai DMSO konsantrasyonu muhafaza edin. nihai ilaç CO elde etmek için hücre kültürü ortamı ile DOX-PA misel seyreltin0.1 uM ncentrations sırasıyla 0.5 uM, 2 uM, 5 uM, 10 uM,. Boş hücre kültürü ortamında bir denetimi kullanın. </li><li> inkübatör 96 oyuklu hücre kültürü plakasının çıkarın ve (her bir grup için n = 4) adım 3.3.5 hazırlandı, farklı işleme maddeleri içeren ortama, 100 ul hücre kültür ortamı yerine. (2, 37 ° C,% 5 CO) ek 72 saat için hücre kültürü inkübatöründe inkübe hücreleri. </li><li> orta aspire ve 3- (4,5-dimetil-tiazol-2-il) ve 0.5 mg / ml ihtiva eden ortam 100 ul -2,5-difenil tetrazolyum bromid (MTT) ilave edin. </li><li> 2 saat daha, hücre kültürü inkübatöründe inkübe hücreleri. Dikkatle orta kaldırmak ve formazan kristalleri eritmek için DMSO 100 ul ekleyin. </li><li> 570 nm&#39;lik bir dalga boyu ve 670 nm&#39;lik bir referans dalga boyu mikroplaka spektrofotometresiyle absorbansı ölçülür. </li><li> Aşağıdaki denklem kullanılarak hücre sağ kalabilirliğinin hesaplanması: <strong&gt; Hücre Canlılığı (%) = (A Testi / bir kontrol) x% 100 NOT: varyans (ANOVA) istatistik testi, tek-yönlü analizi kullanılarak farklı gruplar arasında hücre canlılığı karşılaştır. Ilaç konsantrasyon verileri vs hücre canlılığı dayalı IC 50 hesaplayın. </li></ol></li></ol>

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The authors have nothing to disclose.

Bu çalışmada, miseller hazırlanması için basit ve hızlı bir film-dispersiyon bir yöntem açıklanmaktadır. Bu yöntem, bir amfifilik polimerin kendinden montaj özelliklerinden istifade (örneğin, DSPE-PEG) sulu bir ortamda çekirdek-kabuk yapılı miseller oluşturmak için. Bu misel hazırlama yöntemi birçok avantaja sahiptir. 1. Genellikle lipozom, nanopartiküller ve nanoemülsiyonlar. 19 2 hazırlanmasında kullanılan (örneğin, haddeleme ya da homojenizasyon gibi) karışık büy...

Kemoterapi genellikle çeşitli kanser tiplerini tedavi etmek için kullanılır. hepsi değilse de çoğu, kemoterapi ilaçları daha fazla yaşamı tehdit eden koşullara, bulantı ve ishal gibi yönetilebilir küçük koşullar değişebilir toksik yan etkileri vardır. En antikanser ilaçların toksik olduğundan, normal dokuya bu ilaçların seçici olmayan maruz kaçınılmaz toksisiteye neden olur. Bu nedenle, seçici bir şekilde kanser hücrelerine ilaç teslim bir terapötik yaklaşım için büyük bir ihtiyaç vardır. antikanser ilaçların verilmesi ile diğer zorluk da kötü su çözünürlüğü olduğunu. Genellikle, çözündürücü maddeler, bu az çözünen ilaçların formüle edilmesi gereklidir. Bununla birlikte, dimetil sülfoksit gibi en çözündürücü maddeler, (DMSO), Cremophor EL, ve polisorbat 80 (Tween 80), karaciğer ve böbrek toksisitesi, hemoliz, akut yüksek tansiyon tepkilerine ve periferal nöropatiler neden olabilir. 1 Bu nedenle, güvenli ve biyo-uyumlu formülasyonlar için gerekli olan yoksul klinik kullanımıly çözünür antikanser ilaçlar. Nanocarriers yukarıdaki zorlukları ele almak için ilaç taşıyıcı sistemleri umut vericidir. Bu nanocarriers lipozomlar, 2 nano-tanecikleri, 3 miseller, 4-7, polimer-ilaç konjügatları, 8 ve inorganik malzemeler bulunmaktadır. 9 Çeşitli Nanomedicine ürünleri (örneğin, Doxil, Abraxane ve Genexol) kanser hastalarının tedavisinde düzenleyici kurumlar tarafından onaylanmıştır. 10 Polimerik miseller başarılı antikanser ilaçların verilmesi için kullanılan nano ölçekli ilaç verme taşıyıcılar, umut vericidir. 4-7,11,12 tipik bir polimerik miseller kendinden montaj işlemi boyunca amfifil polimerlerle hazırlanır. Çekirdek-kabuk yapılı polimer miseller bir hidrofilik ve bir hidrofobik kabuk çekirdek bulunmaktadır. hidrofil kabuk sterik miseller stabilize ve kanda kendi dolaşımını uzatabilir. hidrofobik çekirdek etkin bir şekilde hidrofobik d sarabilirizkilim. Çünkü miseller (tipik olarak en az 200 nm) ve uzun sirkülasyon özelliklerinin küçük boyutu, polimerik miseller gelişmiş geçirgenlik ve tutma (EPR) etkileri (pasif tümör hedefleme) ile hedef tümör elde inanılmaktadır. İlaç yükü stabilite miseller yeteneğini hedef tümör için önemlidir. Optimal tümör hedefleme ulaşmak için, misel tümör sitesine ulaşmadan önce en az ilaç sızıntısı, henüz hızlı bir şekilde kanser hücrelerini girdikten sonra ilacı tahliye edilmelidir. formülasyon istikrar ürün geliştirme fizibilite yanı sıra geliştirilen ürünlerin raf ömrünü belirler, çünkü ek olarak, formülasyon stabilitesi, aynı zamanda ürün geliştirme için temel bir gerekliliktir. Son zamanlarda, çok çaba dağıtım taşıyıcılara ilaçların yükleme geliştirmek için yapılmıştır. Lipofilik ön-ilaç bir yaklaşım lipit nanopartiküller ve emülsiyonlar halinde ilaç yüklemesine geliştirmek için araştırılmaktadır bir stratejidir. 13,14 conjilaçlarla lipidlerin ugation önemli ölçüde iyileştirmek ve lipofilliğini nanocarriers lipofilik bileşenleri yükleme ve tutma artırabilir. Burada, lipofilik doksorubisin yanlısı ilaç yüklü miseller hazırlamak için bir protokol açıklar. İlk olarak, lipofilik yanlısı ilaç doksorubisin için sentez prosedürü tarif edilmektedir. Daha sonra, bir film-dispersiyon yöntemi ile miseller oluşturmak için bir protokol verilmektedir. Bu yöntem, başarılı bir şekilde daha önceki çalışmalarda kullanılmıştır. 5 DSPE-PEG başarıyla misel ilaç verilmesi için kullanılmıştır, çünkü miseller hazırlanması için taşıyıcı malzeme olarak seçilmiştir. 15,16 Son olarak, misel karakterize etmek için kullanılan çeşitli in vitro analizler açıklanmaktadır formülasyonlar ve antikanser aktivitesini değerlendirmek için.

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			<td style="width:191px;">Varian, Inc</td>
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			<td>300 NMR&#160;</td>
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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.

Şekil 1, DOX-PS sentez şemasını göstermektedir. DOX-PA pH&#39;a duyarlı hidrazon bağ yoluyla doksorubisin palmitik asit konjügasyonu sentezlendi. palmitik asit hidrazid hafif bir fazlalığı reaksiyon tamamlanmasını sağlamak için kullanılmıştır. Bu reaksiyon yöntemi çok yüksek bir etkinliğe sahip ve doksorubisinin sadece küçük bir miktar, bir 18 saat sonra (Şekil 2) ile gösterilmektedir. Verim yaklaşık% 88 idi. Reaksiyonun son...

Watch this Scientific Journal Video about Preparation and Characterization of Lipophilic Doxorubicin Pro-drug Micelles at JoVE.com

Preparation and Characterization of Lipophilic Doxorubicin Pro-drug Micelles

Lipofilik doksorubisin ön-ilacın hazırlanması ve tanımlanması için bir protokol, 1,2-distearoil, sn -glisero-3-phosphoethanolamine- N yüklü - [amino (polietilen glikol) -2000] (DSPE-PEG) miseller anlatılmıştır.

Lipofilik Doksorubisin Pro-ilaç misellerin Hazırlanması ve Karakterizasyonu

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

preparation-characterization-lipophilic-doxorubicin-pro-drug

Research

JoVE Journal

Bioengineering

14.8K Görüntüleme.  Hampton University.  Lipofilik doksorubisin ön-ilacın hazırlanması ve tanımlanması için bir protokol, 1,2-distearoil, sn -glisero-3-phosphoethanolamine- N yüklü - [amino (polietilen glikol) -2000] (DSPE-PEG) miseller anlatılmıştır. 

Video: Preparation and Characterization of Lipophilic Doxorubicin Pro-drug Micelles

Construction and Characterization of a Novel Vocal Fold Bioreactor

In vitro engineering of mechanically active tissues requires the presentation of physiologically relevant mechanical conditions to cultured cells. To emulate the dynamic environment of vocal folds, a novel vocal fold bioreactor capable of producing vibratory stimulations at fundamental phonation frequencies is constructed and characterized. The device is composed of a function generator, a power amplifier, a speaker selector and parallel vibration chambers. Individual vibration chambers are created by sandwiching a custom-made silicone membrane between a pair of acrylic blocks. The silicone membrane not only serves as the bottom of the chamber but also provides a mechanism for securing the cell-laden scaffold. Vibration signals, generated by a speaker mounted underneath the bottom acrylic block, are transmitted to the membrane aerodynamically by the oscillating air. Eight identical vibration modules, fixed on two stationary metal bars, are housed in an anti-humidity chamber for long-term operation in a cell culture incubator. The vibration characteristics of the vocal fold bioreactor are analyzed non-destructively using a Laser Doppler Vibrometer (LDV). The utility of the dynamic culture device is demonstrated by culturing cellular constructs in the presence of 200-Hz sinusoidal vibrations with a mid-membrane displacement of 40 &#181;m. Mesenchymal stem cells cultured in the bioreactor respond to the vibratory signals by altering the synthesis and degradation of vocal fold-relevant, extracellular matrix components. The novel bioreactor system presented herein offers an excellent in vitro platform for studying vibration-induced mechanotransduction and for the engineering of functional vocal fold tissues.

A novel vocal fold bioreactor capable of delivering physiologically relevant, vibratory stimulation to cultured cells is constructed and characterized. This dynamic culture device, when combined with a fibrous poly(&#949;-caprolactone) scaffold, creates a vocal fold-mimetic environment that modulates the behaviors of mesenchymal stem cells.

Bir Roman Vokal Fold Bioreactor İnşaatı ve Karakterizasyonu

In vitro engineering of mechanically active tissues requires the presentation of physiologically relevant mechanical conditions to cultured cells. To ...

Biyomühendislik

Preparation and Characterization of Novel HDL-mimicking Nanoparticles for Nerve Growth Factor Encapsulation

The objective of this article is to introduce preparation and characterization methods for nerve growth factor (NGF)-loaded, high-density, lipoprotein (HDL)-mimicking nanoparticles (NPs). HDLs are endogenous NPs and have been explored as vehicles for the delivery of therapeutic agents. Various methods have been developed to prepare HDL-mimicking NPs. However, they are generally complicated, time consuming, and difficult for industrial scale-up. In this study, one-step homogenization was used to mix the excipients and form the prototype NPs. NGF is a water-soluble protein of 26 kDa. To facilitate the encapsulation of NGF into the lipid environment of HDL-mimicking NPs, protamine USP was used to form an ion-pair complex with NGF to neutralize the charges on the NGF surface. The NGF/protamine complex was then introduced into the prototype NPs. Apolipoprotein A-I was finally coated on the surface of the NPs. NGF HDL-mimicking NPs showed preferable properties in terms of particle size, size distribution, entrapment efficiency, in vitro release, bioactivity, and biodistribution. With the careful design and exploration of homogenization in HDL-mimicking NPs, the procedure was greatly simplified, and the NPs were made scalable. Moreover, various challenges, such as separating unloaded NGF from the NPs, conducting reliable in vitro release studies, and measuring the bioactivity of the NPs, were overcome.

Simple homogenization was used to prepare novel, high-density, lipoprotein-mimicking nanoparticles to encapsulate nerve growth factor. Challenges, detailed protocols for nanoparticle preparation, in vitro characterization, and in vivo studies are described in this article.

Sinir Büyümesi Faktörü Kapsüllemesi için Yeni HDL&#39;yi taklit eden Nanopartiküllerin Hazırlanması ve Karakterizasyonu

The objective of this article is to introduce preparation and characterization methods for nerve growth factor (NGF)-loaded, high-density, lipoprotein ...

Synthesis of Monocyte-targeting Peptide Amphiphile Micelles for Imaging of Atherosclerosis

Atherosclerosis is a major contributor to cardiovascular disease, the leading cause of death worldwide, which claims 17.3 million lives annually. Atherosclerosis is also the leading cause of sudden death and myocardial infarction, instigated by unstable plaques that rupture and occlude the blood vessel without warning. Current imaging modalities cannot differentiate between stable and unstable plaques that rupture. Peptide amphiphiles micelles (PAMs) can overcome this drawback as they can be modified with a variety of targeting moieties that bind specifically to diseased tissue. Monocytes have been shown to be early markers of atherosclerosis, while large accumulation of monocytes is associated with plaques prone to rupture. Hence, nanoparticles that can target monocytes can be used to discriminate different stages of atherosclerosis. To that end, here, we describe a protocol for the preparation of monocyte-targeting PAMs (monocyte chemoattractant protein-1 (MCP-1) PAMs). MCP-1 PAMs are self-assembled through synthesis under mild conditions to form nanoparticles of 15 nm in diameter with near neutral surface charge. In vitro, PAMs were found to be biocompatible and had a high binding affinity for monocytes. The methods described herein show promise for a wide range of applications in atherosclerosis as well as other inflammatory diseases.

This paper presents a method involving the synthesis and characterization of monocyte-targeting peptide amphiphile micelles and the corresponding assays to test for biocompatibility and ability of the micelle to bind to monocytes.

Monosit hedefleme peptid Amfifil Micelles için düşsel ateroskleroz sentezi

Atherosclerosis is a major contributor to cardiovascular disease, the leading cause of death worldwide, which claims 17.3 million lives annually. ...

Fabrication and Characterization of Optical Tissue Phantoms Containing Macrostructure

The rapid development of new optical imaging techniques is dependent on the availability of low-cost, customizable, and easily reproducible standards. By replicating the imaging environment, costly animal experiments to validate a technique may be circumvented. Predicting and optimizing the performance of in vivo and ex vivo imaging techniques requires testing on samples that are optically similar to tissues of interest. Tissue-mimicking optical phantoms provide a standard for evaluation, characterization, or calibration of an optical system. Homogenous polymer optical tissue phantoms are widely used to mimic the optical properties of a specific tissue type within a narrow spectral range. Layered tissues, such as the epidermis and dermis, can be mimicked by simply stacking these homogenous slab phantoms. However, many in vivo imaging techniques are applied to more spatially complex tissue where three dimensional structures, such as blood vessels, airways, or tissue defects, can affect the performance of the imaging system. 
This protocol describes the fabrication of a tissue-mimicking phantom that incorporates three-dimensional structural complexity using material with optical properties of tissue. Look-up tables provide India ink and titanium dioxide recipes for optical absorption and scattering targets. Methods to characterize and tune the material optical properties are described. The phantom fabrication detailed in this article has an internal branching mock airway void; however, the technique can be broadly applied to other tissue or organ structures.

Optical tissue phantoms are essential tools for calibration and characterization of optical imaging systems and validation of theoretical models. This article details a method for phantom fabrication that includes replication of tissue optical properties and three-dimensional tissue structure.

İmalat ve Macrostructure içeren optik doku hayaletler karakterizasyonu

The rapid development of new optical imaging techniques is dependent on the availability of low-cost, customizable, and easily reproducible standards. By ...

Preparation, Purification, and Use of Fatty Acid-containing Liposomes

Liposomes containing single-chain amphiphiles, particularly fatty acids, exhibit distinct properties compared to those containing diacylphospholipids due to the unique chemical properties of these amphiphiles. In particular, fatty acid liposomes enhance dynamic character, due to the relatively high solubility of single-chain amphiphiles. Similarly, liposomes containing free fatty acids are more sensitive to salt and divalent cations, due to the strong interactions between the carboxylic acid head groups and metal ions. Here we illustrate techniques for preparation, purification, and use of liposomes comprised in part or whole of single chain amphiphiles (e.g., oleic acids).

Liposomes containing single-chain amphiphiles, particularly fatty acids, exhibit distinct properties compared to those containing diacylphospholipids due to the unique chemical properties of single chain amphiphiles. Here we describe techniques for the preparation, purification, and use of liposomes comprised in part or whole of these amphiphiles.

Hazırlık, arıtma ve yağ asidi içeren lipozomlar kullanımı

Liposomes containing single-chain amphiphiles, particularly fatty acids, exhibit distinct properties compared to those containing diacylphospholipids due ...

Detection of Endotoxin in Nano-formulations Using Limulus Amoebocyte Lysate (LAL) Assays

When present in pharmaceutical products, a Gram-negative bacterial cell wall component endotoxin (often also called lipopolysaccharide) can cause inflammation, fever, hypo- or hypertension, and, in extreme cases, can lead to tissue and organ damage that may become fatal. The amounts of endotoxin in pharmaceutical products, therefore, are strictly regulated. Among the methods available for endotoxin detection and quantification, the Limulus Amoebocyte Lysate (LAL) assay is commonly used worldwide. While any pharmaceutical product can interfere with the LAL assay, nano-formulations represent a particular challenge due to their complexity. The purpose of this paper is to provide a practical guide to researchers inexperienced in estimating endotoxins in engineered nanomaterials and nanoparticle-formulated drugs. Herein, practical recommendations for performing three LAL formats including turbidity, chromogenic and gel-clot assays are discussed. These assays can be used to determine endotoxin contamination in nanotechnology-based drug products, vaccines, and adjuvants.

Detection of Endotoxin in Nano-formulations Using Limulus Amoebocyte Lysate LAL Assays

Detection of endotoxins in engineered nanomaterials represents one of the grand challenges in the field of nanomedicine. Here, we present a case study that describes the framework composed of three different LAL formats to estimate potential endotoxin contamination in nanoparticles.

Nano-formülasyonları endotoksin tespiti Limulus Amoebocyte Lysate (LAL) deneyleri kullanarak

When present in pharmaceutical products, a Gram-negative bacterial cell wall component endotoxin (often also called lipopolysaccharide) can cause ...

Growth and Characterization of Irradiated Organoids from Mammary Glands

Organoids derived from the digested tissue are multicellular three-dimensional (3D) constructs that better recapitulate in vivo conditions than cell monolayers. Although they cannot completely model in vivo complexity, they retain some functionality of the original organ. In cancer models, organoids are commonly used to study tumor cell invasion. This protocol aims to develop and characterize organoids from the normal and irradiated mouse mammary gland tissue to evaluate the radiation response in normal tissues. These organoids can be applied to future in vitro cancer studies to evaluate tumor cell interactions with irradiated organoids. Mammary glands were resected, irradiated to 20 Gy and digested in a collagenase VIII solution. Epithelial organoids were separated via centrifugal differentiation, and 3D organoids were developed in 96-well low-adhesion microplates. Organoids expressed the characteristic epithelial marker cytokeratin 14. Macrophage interaction with the organoids was observed in co-culture experiments. This model may be useful for studying tumor-stromal interactions, infiltration of immune cells, and macrophage polarization within an irradiated microenvironment.

Organoids developed from mouse mammary glands were irradiated and characterized to assess epithelial traits and interactions with immune cells. Irradiated organoids can be used to better evaluate cell-cell interactions that may lead to tumor cell recruitment in irradiated normal tissue.

Mammary bezlerinden ışınlanmış Organoidlerin büyüme ve karakterizasyonu

Organoids derived from the digested tissue are multicellular three-dimensional (3D) constructs that better recapitulate in vivo conditions than cell ...

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.

Biyoaktif Hidrofilik Globüler Proteinlerin Tuzakları Için Nanoliposomes Hazırlanması ve Karakterizasyonu

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

Focused Ion Beam Lithography to Etch Nano-architectures into Microelectrodes

With advances in electronics and fabrication technology, intracortical microelectrodes have undergone substantial improvements enabling the production of sophisticated microelectrodes with greater resolution and expanded capabilities. The progress in fabrication technology has supported the development of biomimetic electrodes, which aim to seamlessly integrate into the brain parenchyma, reduce the neuroinflammatory response observed after electrode insertion and improve the quality and longevity of electrophysiological recordings. Here we describe a protocol to employ a biomimetic approach recently classified as nano-architecture. The use of focused ion beam lithography (FIB) was utilized in this protocol to etch specific nano-architecture features into the surface of non-functional and functional single shank intracortical microelectrodes. Etching nano-architectures into the electrode surface indicated possible improvements of biocompatibility and functionality of the implanted device. One of the benefits of using FIB is the ability to etch on manufactured devices, as opposed to during the fabrication of the device, facilitating boundless possibilities to modify numerous medical devices post-manufacturing. The protocol presented herein can be optimized for various material types, nano-architecture features, and types of devices. Augmenting the surface of implanted medical devices can improve the device performance and integration into the tissue.

We have shown that the etching of nano-architecture into intracortical microelectrode devices may reduce the inflammatory response and has the potential to improve electrophysiological recordings. The methods described herein outline an approach to etch nano-architectures into the surface of non-functional and functional single shank silicon intracortical microelectrodes.

Etch Nano mimarilerine mikroelektrotlara odaklanmış iyon ışını Litografisi

With advances in electronics and fabrication technology, intracortical microelectrodes have undergone substantial improvements enabling the production of ...

Generation and Quantitative Characterization of Functional and Polarized Biliary Epithelial Cysts

Cholangiocytes, the epithelial cells that line up the bile ducts in the liver, oversee bile formation and modification. In the last twenty years, in the context of liver diseases, 3-dimensional (3D) models based on cholangiocytes have emerged such as cysts, spheroids, or tube-like structures to mimic tissue topology for organogenesis, disease modeling, and drug screening studies. These structures have been mainly obtained by embedding cholangiocytes in a hydrogel. The main purpose was to study self-organization by addressing epithelial polarity, functional, and morphological properties. However, very few studies focus on cyst formation efficiency. When this is the case, the efficiency is often quantified from images of a single plane. Functional assays and structural analysis are performed without representing the potential heterogeneity of cyst distribution arising from hydrogel polymerization heterogeneities and side effects. Therefore, the quantitative analysis, when done, cannot be used for comparison from one article to another. Moreover, this methodology does not allow comparisons of 3D growth potential of different matrices and cell types. Additionally, there is no mention of the experimental troubleshooting for immunostaining cysts. In this article, we provide a reliable and universal method to show that the initial cell distribution is related to the heterogeneous vertical distribution of cyst formation. Cholangiocyte cells embedded in hydrogel are followed with Z-stacks analysis along the hydrogel depth over the time course of 10 days. With this method, a robust kinetics of cyst formation efficiency and growth is obtained. We also present methods to evaluate cyst polarity and secretory function. Finally, additional tips for optimizing immunostaining protocols are provided in order to limit cyst collapse for imaging. This approach can be applied to other 3D cell culture studies, thus opening the possibilities to compare one system to another.

Three-dimensional (3D) cellular systems are relevant models for investigating organogenesis. A hydrogel-based method for biliary cysts production and their characterization is proposed. This protocol unravels the barriers of 3D characterization, with a straightforward and reliable method to assess cyst formation efficiency, sizes, and to test their functionality.

Fonksiyonel ve Polarize Safra Epitel Kistlerinin Oluşumu ve Kantitatif Karakterizasyonu

Cholangiocytes, the epithelial cells that line up the bile ducts in the liver, oversee bile formation and modification. In the last twenty years, in the ...