この作品は、がん、ナノテクノロジー優秀パイロット助成金、大学のがん研究基金と国立予防衛生研究所K - 12キャリア開発賞のためのキャロライナセンターに対するゴルファーによって賄われていた。

1。 PLGA - B - PEGポリマーの合成<ol><li>ターミナルカルボキシレート基（PLGA -カルボン酸）とポリ（D、L -ラクチド- co -グリコリド）（PLGA）を5mMの濃度で（のような材料のセクションに記載されている）PLGAのための任意の溶媒に溶解させる。 PLGAは、穏やかに攪拌しながらこの濃度で溶解させることができる。 </li><li> NHS（分子量115.09）とEDC（分子量191.7）の両方は、25mMの濃度でPLGA溶液に溶解されています。 （EDCとNHSの両方が、PLGAに比べて5倍の化学量論的過剰量で添加されています）。 PLGA -カルボキシレートは、約1時間穏やかに撹拌しながら、PLGA -カルボン酸液にEDCとNHSを追加することにより、PLGA - NHSに変換されます。 </li><li>反応生成物PLGA - NHSは、洗浄液のメタノールを添加することにより沈殿させる。メタノールの約10倍の量の過剰がソリューションに追加されます。ソリューションをPLGA - NHS外沈殿、2000 × gで遠心分離し、上清を破棄され（EDCとNHSのトレースを削除します。メタノールで洗浄のこの手順では、少なくとも3回繰り返される。 </li><li> PLGA - NHSペレットは、洗浄溶液の痕跡を除去するために30分間真空下で乾燥させる。 </li><li> PLGA - NHSペレットは現在、PLGAを溶解するために最初に使用したのと同じ濃度で同じ溶媒に再溶解している。ヘテロPEG（アミン- PEG -カルボン酸）は、その後、5mMの濃度のPLGA溶液に添加される（1:1の化学量論比）。混合溶液を絶えず撹拌しながら24時間インキュベートされる。 </li><li> 24時間後、反応生成物PLGA - B - PEGブロック共重合体は、過剰の洗浄液のメタノールを添加することにより沈殿させる。三回上記のように洗浄し、遠心分離処理を繰り返します。これは、すべての過剰な未反応のPEGを削除します。 </li><li> PLGA - B - PEGブロック共重合体は、真空下で乾燥させる。 </li></ol> 2。 PLGA - B - PEGナノ粒子の調製表面にPEGで覆われたPLGAのコアを持つナノ粒子は、これらのジブロック共重合体を調製することができる。別の疎水性薬物の様々なそのようなナノ粒子に封入することができる。蛍光化合物がナノ粒子に封入することができますまたはPLGAに結合させることができるため、これらのナノ粒子は、蛍光イメージングに使用することができます。 ナノ析出法は、カプセル化するために必要な貨物は、本質的に疎水性の高い場合は特にナノ粒子を作るために使用されます。 <ol><li> PLGA - B - PEGブロック共重合体と薬/貨物は、（カプセル化する）PLGAを溶解する任意の溶媒に溶解されています。 PLGAは、アセトニトリル、DCM、テトラヒドロフラン、アセトンや酢酸エチルなど、多くの一般的な溶剤で溶解させることができる。それはナノ粒子の性質に影響を与えるとして、溶媒の選択は、重要です。したがって、適当な溶媒は、このステップで使用する必要があります。 </li><li>ポリマー/薬剤混合物を次いで、約3 mg / mLの最終的なポリマーの濃度を与える水を攪拌しながら3〜5ボリュームに滴下する。 （図2） </li><li>攪拌は、ナノ粒子が自己集合によって形成し、有機溶剤の痕跡を除去できるように、減圧下で2時間継続される。 </li><li>収穫と精製：ナノ粒子を洗浄し、アミコンフィルター（MWCO 20KDa）を使用して10分間2700 × gで遠心分離によって濃縮し、PBSに再構成されています。これは、すべての非捕捉された薬剤/貨物を削除します。このようなサイズ、表面電荷、および薬物充填効率などの基本的な生物物理学的特性評価は、より良いナノ粒子の特性を理解するために実行することができます。 </li></ol> 3。ストレージ凍結乾燥は、ナノ粒子11を格納するために一般的に使用される方法です。凍結乾燥は、長期安定性12ナノ粒子の物理的および化学的特性を保持します。凍結乾燥プロセスは、粒子へのストレスの原因と処方を不安定にすることができます、凍結保護剤（凍結​​ストレスからの保護）とlyo -保護剤を（乾燥ストレスからの保護）一般的に使用されるように。これらの保護剤の選択は、蓄積時間13の所望の長さによって決定されます。 <ol><li>凍結乾燥では、そのTg以下に凍結することにより、サンプルの総凝固があります。 </li><li>乾燥工程では、氷が昇華によって除去される。温度と圧力は、効率的な凍結乾燥のプロセスを達成するために最適化する必要があります。 </li></ol> 4。代表的な結果： PLGA - B - PEGジブロック共重合体のキャラクタリゼーションさまざまな手法は、ポリマーの成功共役を確認するために使用することができます。 PLGA - B - PEGの組成は、400 MHzの1H核磁気共鳴（NMR）を用いて特徴づけることができる。形成された生成物（PLGA - B - PEG）の分子量はゲルパーミエーションクロマトグラフィー（GPC）によって確認することができます。 PLGA - B - PEG分子量distribution個の曲線と溶出時間は、PLGAとPEG単独で異なっている必要があります。組み合わせでは、これらの技術は、形成された製品を特徴づけ、抱合反応が成功したかどうかを判断する必要があります。 PLGA - B - PEGナノ粒子のキャラクタリゼーション粒径と粒度分布は動的光散乱法によって測定することができる。ナノ析出プロセスのさまざまなパラメータは、粒子の大きさに影響を与えます。最初に使用されるポリマー（PLGAとPEGの両方）の分子量は、また粒度分布に影響を与えます。遷移電子顕微鏡（TEM）も、図3に示すようにナノ粒子のサイズ分布と構造を確認するために使用することができます。粒径の範囲は、nmの範囲である。不均一なサイズ分布を持つ大規模な粒子サイズは、抱合反応やナノ析出法のニーズの最適化でエラーを示している可能性があります。さらに、表面のゼータ電位はZetaPALSによって測定することができる。 薬/貨物積載効率は標準HPLCで定量することができる。 粒子は、有機溶剤及びHPLCに溶解させる薬剤/貨物（図4）の吸光度を測定するために実行することができます。ナノ粒子の固定量を30スライド- A - Lyzer MINI透析ユニットで透析されている既知の場所薬物放出動力学的研究を行うことができます。一定の時間間隔で、透析ユニット内のコンテンツが収集され、有機溶剤の等量は、ナノ粒子を溶解するために追加されます。 HPLCは、薬物/貨物コンテンツを定量化するために、これらのサンプルに対して行われます。 <img alt="図1" src="/files/ftp_upload/3398/3398fig1.jpg" /> 図1。 EDC / NHS化学 <img alt="図2" src="/files/ftp_upload/3398/3398fig2.jpg" /> 図2。高分子ナノ粒子を調製するためのナノ析出法は、PEG - PLGAジブロックおよび粒子にロードされる薬物や貨物を含む（アセトニトリルまたはDCM）溶剤の有機溶液をH 2 Oを攪拌しながら3〜5 mLを滴下する<img alt="図3" src="/files/ftp_upload/3398/3398fig3.jpg" /> 図3。 nanoparticesの透過型電子顕微鏡。PEG - PLGAはwortaminを含むナノ粒子のTEM像。リンタングステン酸は、造影剤として使用されました。 <img alt="図4" src="/files/ftp_upload/3398/3398fig4.jpg" /> 図4。 PBSで透析後のナノ粒子からのパクリタキセルのナノ粒子からの薬物の放出制御が。リリース。記載時に、粒子は透析カセットから取り出し、アセトニトリルに可溶化。溶液をHPLCで測定した。ナノ粒子の2つの別個のロットを比較した。 <div id="myEventWatcherDiv"></div><div id="myEventWatcherDiv"></div>

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ジブロックコポリマーを用いたナノ析出法は、高分子ナノ粒子を設計するための簡単​​、迅速な方法を表します。得られたナノ粒子は、難溶性化合物の送達に利用することができる疎水性のコアで構成されています。標的リガンドへの潜在的な、さらに結合のための部分を提供しながら、表面親水性層は、優れた水溶性を可能にします。 リポソーム、高分子ナノ粒子?...

<table border="1"><tbody><tr><td> 試薬 </td><td> 会社 </td><td> カタログ番号 </td><td> コメント </td></tr><tr><td> EDC </td><td>サーモサイエンティフィック</td><td> 22980 </td><td>共役試薬</td></tr><tr><td> NHS </td><td>サーモサイエンティフィック</td><td> 24500 </td><td>共役試薬</td></tr><tr><td>アミン- PEG -カルボン酸</td><td>ライサン島バイオ（株） </td><td> NH2 - PEG - CM - 5000 </td><td>ポリマー（任意のPEG MW、5000がここに記載されている使用可能） </td></tr><tr><td> PLGA - carbxylate </td><td> Lactel </td><td> B6013 - 2 </td><td>高分子</td></tr><tr><td>ジクロロメタン（DCM） </td><td>シグマアルドリッチ</td><td> 34856 </td><td>溶剤</td></tr><tr><td>アセトニトリル&gt; 99％純度</td><td>シグマアルドリッチ</td><td> 34851 </td><td>溶剤</td></tr><tr><td>メタノール&gt; 99％純度</td><td>シグマアルドリッチ</td><td> 34860 </td><td>洗う</td></tr></tbody></table>

formulation of diblock polymeric nanoparticles through nanoprecipitation technique

ナノテクノロジーは、活用のスケールでナノメートル（ナノ粒子）の粒子のユニークな特性を含む科学の比較的新しい支店です。ナノ粒子はその大きさ、組成や表面の化学的性質を注意深く制御することができる正確な方法で設計することができる。これは、溶解性、拡散性、体内分布、放出特性と免疫原性のようなそれらの貨物の基本的なプロパティの一部を変更するために前例のない自由を可能にします。世の中に出て以来、ナノ粒子は、薬物送達、イメージング、および細胞生物学1から4を含む科学や医学の多くの分野で利用されている。しかし、それは完全に知覚される技術的な障壁のために&quot;ナノテクノロジー研究所&quot;の外で利用されていません。この記事では、我々は潜在的なアプリケーションの広い範囲を有するポリマーベースのナノ粒子のプラットフォームを合成する簡単な方法を説明します。 最初のステップは、疎水性ドメインと親水性ドメインの両方を持つジブロック共重合体を合成することである。モデルポリマーとしてPLGAとPEGを使用して、我々は、EDC / NHS化学5（図1）を使用して結合反応を説明した。我々はまた、ポリマーの精製のプロセスについて説明します。合成したジブロックコポリマーは、疎水性 - 親水性相互作用を通してナノ析出プロセスでナノ粒子に自己集合することができます。 に記載のポリマーナノ粒子は、非常に汎用性があります。ナノ粒子の疎水性コアは、薬物送達experiments6に対して難溶性薬物を運ぶために利用することができます。さらに、ナノ粒子は、DMSOのような溶剤を必要とするワートマニン、などの難溶性分子生物学試薬、のために有毒な溶剤の問題を克服することができます。しかし、DMSOは細胞毒性が、実験の妨げになることがあります。これらの難溶性の薬物や試薬は、効果的に最小限の毒性を有するポリマーナノ粒子を使用して配信することができます。ポリマーナノ粒子はまた、蛍光色素でロードされ、細胞内輸送の研究に利用することができます。最後に、これらのポリマーナノ粒子は、表面のPEGを介してリガンドを標的に結合させることができる。そのような標的ナノ粒子は,7 - 10や細胞内の特定のエピトープにラベルを付けるために利用することができます。

Watch this Scientific Journal Video about Formulation of Diblock Polymeric Nanoparticles through Nanoprecipitation Technique at JoVE.com

Formulation of Diblock Polymeric Nanoparticles through Nanoprecipitation Technique

この記事では、ジブロック共重合体を用いたポリマーをベースとしたナノ粒子を合成するナノ析出の方法を説明します。私たちは、ジブロックの合成共重合体、ナノ析出技術、及び潜在的なアプリケーションについて説明します。

ナノ析出法を通じて、ジブロック高分子ナノ粒子の製剤

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

formulation-diblock-polymeric-nanoparticles-through-nanoprecipitation

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37.1K ビュー.  University of North Carolina School of Medicine. この記事では、ジブロック共重合体を用いたポリマーをベースとしたナノ粒子を合成するナノ析出の方法を説明します。私たちは、ジブロックの合成共重合体、ナノ析出技術、及び潜在的なアプリケーションについて説明します。

ビデオ: Formulation of Diblock Polymeric Nanoparticles through Nanoprecipitation Technique

Evaluation of Polymeric Gene Delivery Nanoparticles by Nanoparticle Tracking Analysis and High-throughput Flow Cytometry

Non-viral gene delivery using polymeric nanoparticles has emerged as an attractive approach for gene therapy to treat genetic diseases1 and as a technology for regenerative medicine2. Unlike viruses, which have significant safety issues, polymeric nanoparticles can be designed to be non-toxic, non-immunogenic, non-mutagenic, easier to synthesize, chemically versatile, capable of carrying larger nucleic acid cargo and biodegradable and/or environmentally responsive. Cationic polymers self-assemble with negatively charged DNA via electrostatic interaction to form complexes on the order of 100 nm that are commonly termed polymeric nanoparticles. Examples of biomaterials used to form nanoscale polycationic gene delivery nanoparticles include polylysine, polyphosphoesters, poly(amidoamines)s and polyethylenimine (PEI), which is a non-degradable off-the-shelf cationic polymer commonly used for nucleic acid delivery1,3 . Poly(beta-amino ester)s (PBAEs) are a newer class of cationic polymers4 that are hydrolytically degradable5,6 and have been shown to be effective at gene delivery to hard-to-transfect cell types such as human retinal endothelial cells (HRECs)7, mouse mammary epithelial cells8, human brain cancer cells9 and macrovascular (human umbilical vein, HUVECs) endothelial cells10.
A new protocol to characterize polymeric nanoparticles utilizing nanoparticle tracking analysis (NTA) is described. In this approach, both the particle size distribution and the distribution of the number of plasmids per particle are obtained11. In addition, a high-throughput 96-well plate transfection assay for rapid screening of the transfection efficacy of polymeric nanoparticles is presented. In this protocol, poly(beta-amino ester)s (PBAEs) are used as model polymers and human retinal endothelial cells (HRECs) are used as model human cells. This protocol can be easily adapted to evaluate any polymeric nanoparticle and any cell type of interest in a multi-well plate format.

A protocol for nanoparticle tracking analysis (NTA) and high-throughput flow cytometry to evaluate polymeric gene delivery nanoparticles is described. NTA is utilized to characterize the nanoparticle particle size distribution and the plasmid per particle distribution. High-throughput flow cytometry enables quantitative transfection efficacy evaluation for a library of gene delivery biomaterials.

ナノ粒子トラッキング分析とハイスループットフローサイトメトリーによる高分子遺伝子デリバリーナノ粒子の評価

Non-viral gene delivery using polymeric nanoparticles has emerged as an attractive approach for gene therapy to treat genetic diseases1 and as a ...

生物工学

Programming Stem Cells for Therapeutic Angiogenesis Using Biodegradable Polymeric Nanoparticles

Controlled vascular growth is critical for successful tissue regeneration and wound healing, as well as for treating ischemic diseases such as stroke, heart attack or peripheral arterial diseases. Direct delivery of angiogenic growth factors has the potential to stimulate new blood vessel growth, but is often associated with limitations such as lack of targeting and short half-life in vivo. Gene therapy offers an alternative approach by delivering genes encoding angiogenic factors, but often requires using virus, and is limited by safety concerns. Here we describe a recently developed strategy for stimulating vascular growth by programming stem cells to overexpress angiogenic factors in situ using biodegradable polymeric nanoparticles. Specifically our strategy utilized stem cells as delivery vehicles by taking advantage of their ability to migrate toward ischemic tissues in vivo. Using the optimized polymeric vectors, adipose-derived stem cells were modified to overexpress an angiogenic gene encoding vascular endothelial growth factor (VEGF). We described the processes for polymer synthesis, nanoparticle formation, transfecting stem cells in vitro, as well as methods for validating the efficacy of VEGF-expressing stem cells for promoting angiogenesis in a murine hindlimb ischemia model.

We describe the method of programming stem cells to overexpress therapeutic factors for angiogenesis using biodegradable polymeric nanoparticles. Processes described include polymer synthesis, transfecting adipose-derived stem cells in vitro, and validating the efficacy of programmed stem cells to promote angiogenesis in a murine hindlimb ischemia model.

生分解性ポリマーナノ粒子を用いた治療的血管形成のためのプログラミング幹細胞

Controlled vascular growth is critical for successful tissue regeneration and wound healing, as well as for treating ischemic diseases such as stroke, ...

Microfluidic Fabrication of Polymeric and Biohybrid Fibers with Predesigned Size and Shape

A &#8220;sheath&#8221; fluid passing through a microfluidic channel at low Reynolds number can be directed around another &#8220;core&#8221; stream and used to dictate the shape as well as the diameter of a core stream.&#160;Grooves in the top and bottom of a microfluidic channel were designed to direct the sheath fluid and shape the core fluid.&#160;By matching the viscosity and hydrophilicity of the sheath and core fluids, the interfacial effects are minimized and complex fluid shapes can be formed.&#160;Controlling the relative flow rates of the sheath and core fluids determines the cross-sectional area of the core fluid.&#160;Fibers have been produced with sizes ranging from 300 nm to ~1 mm, and fiber cross-sections can be round, flat, square, or complex as in the case with double anchor fibers. Polymerization of the core fluid downstream from the shaping region solidifies the fibers.&#160;Photoinitiated click chemistries are well suited for rapid polymerization of the core fluid by irradiation with ultraviolet light.&#160;Fibers with a wide variety of shapes have been produced from a list of polymers including liquid crystals, poly(methylmethacrylate), thiol-ene and thiol-yne resins, polyethylene glycol, and hydrogel derivatives. Minimal shear during the shaping process and mild polymerization conditions also makes the fabrication process well suited for encapsulation of cells and other biological components.

Two adjacent fluids passing through a grooved microfluidic channel can be directed to form a sheath around a prepolymer core; thereby&#160;determining both shape and cross-section. Photoinitiated polymerization, such as thiol click chemistry, is well suited for rapidly solidifying the core fluid into a microfiber with predetermined size and shape.

あらかじめ設計されたサイズと形状を用いるポリマー繊維およびバイオハイブリッド繊維のマイクロ流体製造

A &#8220;sheath&#8221; fluid passing through a microfluidic channel at low Reynolds number can be directed around another &#8220;core&#8221; stream and ...

Polymeric Microneedle Array Fabrication by Photolithography

This manuscript describes the fabrication of polymeric microneedle (MN) arrays by photolithography. It involves a simple mold-free process by using a photomask consisting of embedded micro-lenses. Embedded micro-lenses were found to influence MN geometry (sharpness). Robust MN arrays with tip diameters ranging between 41.5 &#181;m &#177; 8.4 &#181;m and 71.6 &#181;m &#177; 13.7 &#181;m, with two different lengths (1,336 &#181;m &#177; 193 &#181;m and 957 &#181;m &#177; 171 &#181;m) were fabricated. These MN arrays may provide potential applications in delivery of low molecular and macromolecular therapeutic agents through skin.

Here, we present a protocol describing a mold-free fabrication process of the polymeric microneedles by photolithography.

フォトリソグラフィ法により高分子マイクロニードルアレイの作製

This manuscript describes the fabrication of polymeric microneedle (MN) arrays by photolithography. It involves a simple mold-free process by using a ...

Fabricating Superhydrophobic Polymeric Materials for Biomedical Applications

Superhydrophobic materials, with surfaces possessing permanent or metastable non-wetted states, are of interest for a number of biomedical and industrial applications. Here we describe how electrospinning or electrospraying a polymer mixture containing a biodegradable, biocompatible aliphatic polyester (e.g., polycaprolactone and poly(lactide-co-glycolide)), as the major component, doped with a hydrophobic copolymer composed of the polyester and a stearate-modified poly(glycerol carbonate) affords a superhydrophobic biomaterial. The fabrication techniques of electrospinning or electrospraying provide the enhanced surface roughness and porosity on and within the fibers or the particles, respectively. The use of a low surface energy copolymer dopant that blends with the polyester and can be stably electrospun or electrosprayed affords these superhydrophobic materials. Important parameters such as fiber size, copolymer dopant composition and/or concentration, and their effects on wettability are discussed. This combination of polymer chemistry and process engineering affords a versatile approach to develop application-specific materials using scalable techniques, which are likely generalizable to a wider class of polymers for a variety of applications.

Two- and three-dimensional superhydrophobic polymeric materials are prepared by electrospinning or electrospraying biodegradable polymers blended with a lower surface energy polymer of similar composition.

バイオメディカル応用のための超疎水性高分子材料を製造します

Superhydrophobic materials, with surfaces possessing permanent or metastable non-wetted states, are of interest for a number of biomedical and industrial ...

Predicting Gene Silencing Through the Spatiotemporal Control of siRNA Release from Photo-responsive Polymeric Nanocarriers

New materials and methods are needed to better control the binding vs. release of nucleic acids for a wide range of applications that require the precise regulation of gene activity. In particular, novel stimuli-responsive materials with improved spatiotemporal control over gene expression would unlock translatable platforms in drug discovery and regenerative medicine technologies. Furthermore, an enhanced ability to control nucleic acid release from materials would enable the development of streamlined methods to predict nanocarrier efficacy a priori, leading to expedited screening of delivery vehicles. Herein, we present a protocol for predicting gene silencing efficiencies and achieving spatiotemporal control over gene expression through a modular photo-responsive nanocarrier system. Small interfering RNA (siRNA) is complexed with mPEG-b-poly(5-(3-(amino)propoxy)-2-nitrobenzyl methacrylate) (mPEG-b-P(APNBMA)) polymers to form stable nanocarriers that can be controlled with light to facilitate tunable, on/off siRNA release. We outline two complementary assays employing fluorescence correlation spectroscopy and gel electrophoresis for the accurate quantification of siRNA release from solutions mimicking intracellular environments. Information gained from these assays was incorporated into a simple RNA interference (RNAi) kinetic model to predict the dynamic silencing responses to various photo-stimulus conditions. In turn, these optimized irradiation conditions allowed refinement of a new protocol for spatiotemporally controlling gene silencing. This method can generate cellular patterns in gene expression with cell-to-cell resolution and no detectable off-target effects. Taken together, our approach offers an easy-to-use method for predicting dynamic changes in gene expression and precisely controlling siRNA activity in space and time. This set of assays can be readily adapted to test a wide variety of other stimuli-responsive systems in order to address key challenges pertinent to a multitude of applications in biomedical research and medicine.

We present a novel method that uses photo-responsive block copolymers for more efficient spatiotemporal control of gene silencing with no detectable off-target effects. Additionally, changes in gene expression can be predicted using straightforward siRNA release assays and simple kinetic modeling.

光応答性高分子ナノキャリアからのsiRNA放出の時空間制御による遺伝子サイレンシングの予測

New materials and methods are needed to better control the binding vs. release of nucleic acids for a wide range of applications that require the precise ...

Rapid, Scalable Assembly and Loading of Bioactive Proteins and Immunostimulants into Diverse Synthetic Nanocarriers Via Flash Nanoprecipitation

Nanomaterials present a wide range of options to customize the controlled delivery of single and combined molecular payloads for therapeutic and imaging applications. This increased specificity can have significant clinical implications, including decreased side effects and lower dosages with higher potency. Furthermore, the in situ targeting and controlled modulation of specific cell subsets can enhance in vitro and in vivo investigations of basic biological phenomena and probe cell function. Unfortunately, the required expertise in nanoscale science, chemistry and engineering often prohibit laboratories without experience in these fields from fabricating and customizing nanomaterials as tools for their investigations or vehicles for their therapeutic strategies. Here, we provide protocols for the synthesis and scalable assembly of a versatile non-toxic block copolymer system amenable to the facile formation and loading of nanoscale vehicles for biomedical applications. Flash nanoprecipitation is presented as a methodology for rapid fabrication of diverse nanocarriers from poly(ethylene glycol)-bl-poly(propylene sulfide) copolymers. These protocols will allow laboratories with a wide range of expertise and resources to easily and reproducibly fabricate advanced nanocarrier delivery systems for their applications. The design and construction of an automated instrument that employs a high-speed syringe pump to facilitate the flash nanoprecipitation process and to allow enhanced control over the homogeneity, size, morphology and loading of polymersome nanocarriers is described.

Nanomaterials provide versatile mechanisms of controlled therapeutic delivery for both basic science and translational applications, but their fabrication often requires expertise that is unavailable in most biomedical laboratories. Here, we present protocols for the scalable fabrication and therapeutic loading of diverse self-assembled nanocarriers using flash nanoprecipitation.

高速でスケーラブルなアセンブリおよびフラッシュ Nanoprecipitation を介して多様な合成ナノキャリアへ生理活性タンパク質や生理の読み込み

Nanomaterials present a wide range of options to customize the controlled delivery of single and combined molecular payloads for therapeutic and imaging ...

Protein Kinase C-delta Inhibitor Peptide Formulation using Gold Nanoparticles

Protein kinase C-delta inhibitor (PKC&#948;i) is a promising drug to prevent ischemia-reperfusion-induced organ injury. It is usually conjugated to a cell-penetrating peptide, TAT, for intracellular delivery. However, TAT has shown non-specific biological activities. Gold nanoparticles (GNPs) can be used as drug delivery carriers without recognized toxicity. Therefore, we have used a GNP/peptide hybrid to deliver PKC&#948;i. Two short peptides (P2: CAAAAE and P4: CAAAAW), at a 95:5 ratio, were used to modify the surface properties of GNP. GNPs conjugated with PKC&#948;i (GNP/PKCi) are stable in distilled water, 0.9% NaCl, and phosphate-buffered saline (PBS) containing bovine serum albumin or fetal bovine serum. Intravenous injection of GNP-PKCi was previously shown to prevent ischemia-reperfusion injury of the lung. This article outlines a protocol to formulate GNP/PKCi and assess the physiochemical properties of GNP/PKCi. We have used similar methods to formulate other peptide-based drugs with GNP. This article will hopefully draw more attention to this novel intracellular drug delivery technology and its applications in vivo.

We have previously used a gold nanoparticle peptide hybrid to intravenously deliver a synthetic peptide, protein kinase C-delta inhibitor, which reduced ischemia-reperfusion-induced acute lung injury. Here we show the detailed protocol of the drug formulation. Other intracellular peptides can be formulated similarly.

金ナノ粒子を用いた蛋白質キナーゼ C デルタ阻害ペプチド製剤

Protein kinase C-delta inhibitor (PKC&#948;i) is a promising drug to prevent ischemia-reperfusion-induced organ injury. It is usually conjugated to a ...

Flash NanoPrecipitation for the Encapsulation of Hydrophobic and Hydrophilic Compounds in Polymeric Nanoparticles

The formulation of a therapeutic compound into nanoparticles (NPs) can impart unique properties. For poorly water-soluble drugs, NP formulations can improve bioavailability and modify drug distribution within the body. For hydrophilic drugs like peptides or proteins, encapsulation within NPs can also provide protection from natural clearance mechanisms. There are few techniques for the production of polymeric NPs that are scalable. Flash NanoPrecipitation (FNP) is a process that uses engineered mixing geometries to produce NPs with narrow size distributions and tunable sizes between 30 and 400 nm. This protocol provides instructions on the laboratory-scale production of core-shell polymeric nanoparticles of a target size using FNP. The protocol can be implemented to encapsulate either hydrophilic or hydrophobic compounds with only minor modifications. The technique can be readily employed in the laboratory at milligram scale to screen formulations. Lead hits can directly be scaled up to gram- and kilogram-scale. As a continuous process, scale-up involves longer mixing process run time rather than translation to new process vessels. NPs produced by FNP are highly loaded with therapeutic, feature a dense stabilizing polymer brush, and have a size reproducibility of &#177; 6%.

Flash NanoPrecipitation (FNP) is a scalable approach to produce polymeric core-shell nanoparticles. Lab-scale formulations for the encapsulation of hydrophobic or hydrophilic therapeutics are described.

高分子ナノ粒子の疎水性および親水性化合物のカプセル化のため NanoPrecipitation をフラッシュします。

The formulation of a therapeutic compound into nanoparticles (NPs) can impart unique properties. For poorly water-soluble drugs, NP formulations can ...

Preparation of Neutrally-charged, pH-responsive Polymeric Nanoparticles for Cytosolic siRNA Delivery

The success of siRNA as a targeted molecular medicine is dependent upon its efficient cytosolic delivery to cells within the tissue of pathology. Clinical success for treating previously &#8216;undruggable&#8217; hepatic disease targets with siRNA has been achieved. However, efficient tumor siRNA delivery necessitates additional pharmacokinetic design considerations, including long circulation time, evasion of clearance organs (e.g., liver and kidneys), and tumor penetration and retention. Here, we describe the preparation and in vitro physicochemical/biological characterization of polymeric nanoparticles designed for efficient siRNA delivery, particularly to non-hepatic tissues such as tumors. The siRNA nanoparticles are prepared by electrostatic complexation of siRNA and the diblock copolymer poly(ethylene glycol-b-[2-(dimethylamino)ethyl methacrylate-co-butyl methacrylate]) (PEG-DB) to form polyion complexes (polyplexes) where siRNA is sequestered within the polyplex core and PEG forms a hydrophilic, neutrally-charged corona. Moreover, the DB block becomes membrane-lytic as vesicles of the endolysosomal pathway acidify (&#60; pH 6.8), triggering endosomal escape and cytosolic delivery of siRNA. Methods to characterize the physicochemical characteristics of siRNA nanoparticles such as size, surface charge, particle morphology, and siRNA loading are described. Bioactivity of siRNA nanoparticles is measured using luciferase as a model gene in a rapid and high-throughput gene silencing assay. Designs which pass these initial tests (such as PEG-DB-based polyplexes) are considered appropriate for translation to preclinical animal studies assessing the delivery of siRNA to tumors or other sites of pathology.

Methods to prepare and characterize the physicochemical properties and bioactivity of neutrally-charged, pH-responsive siRNA nanoparticles are presented. Criteria for successful siRNA nanomedicines such as size, morphology, surface charge, siRNA loading, and gene silencing are discussed.

細胞質 siRNA 送達のための、中立的充電された pH 応答性高分子ナノ粒子の調製

The success of siRNA as a targeted molecular medicine is dependent upon its efficient cytosolic delivery to cells within the tissue of pathology. Clinical ...