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Jun 2020

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Preparation and Characterization of Poly(2-oxazoline) Micelles for the Solubilization and Delivery of Water Insoluble Drugs
用于溶解和输送难溶性药物的聚恶唑啉胶束的制备及特性   

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Abstract

Many new drug development candidates are highly lipophilic compounds with low water solubility. This constitutes a formidable challenge for the use of such compounds for cancer therapy, where high doses and intravenous injections are needed (Di et al., 2012). Here, we present a poly(2-oxazoline) polymer (POx)-based nanoformulation strategy to solubilize and deliver hydrophobic drugs. POx micelles are prepared by a simple thin-film hydration method. In this method, the drug and polymer are dissolved in a common solvent and allowed to mix, following which the solvent is evaporated using mild heating conditions to form a thin film. The micelles form spontaneously upon hydration with saline. POx nanoformulation of hydrophobic drugs is unique in that it has a high drug loading capacity, which is superior to micelles of conventional surfactants. Moreover, multiple active pharmaceutical ingredients (APIs) can be included within the same POx micelle, thereby enabling the codelivery of binary as well as ternary drug combinations (Han et al., 2012; He et al., 2016).

Keywords: Lipophilic (亲脂性的), Poly(2-oxazoline) (聚(2-恶唑啉)), Nanoformulation (纳米制剂), Surfactants (表面活性剂), Active pharmaceutical ingredients (药物活性成分), Polymeric micelles (聚合物胶束)

Background

Recent statistics show that only 3.7% of the new drug candidates that enter clinical testing are approved for use in cancer treatment. This has been primarily attributed to the poor pharmacokinetics of poorly water-soluble drug candidates, which results in suboptimal performance (Gala et al., 2020). POx polymeric micelles offer several advantages over traditional drug delivery systems such as liposomes, microparticles, and nanogels, among others. The unparalleled high solubilizing capacity of POx micelles for a large variety of hydrophobic drugs enables the delivery of greater amounts of drugs with a substantially lesser amount of excipient (Luxenhofer et al., 2010, He et al., 2016). POx-based drug formulations are easy to prepare, safe, and stable. Additionally, a quantitative structure-property relationship (QSPR) model has been developed to predict drug loading into POx micelles, which can be utilized to facilitate high throughput screening of sparingly soluble drug development candidates for incorporation in POx micelles (Alves et al., 2019).


Materials and Reagents

  1. PVC tubing (Nalgene, 1/4” ID )

  2. Eppendorf tubes (Fisher Scientific, catalog number: 05-408-129 )

  3. Pipette tips (Fisher Scientific)

  4. 11 mm plastic autosampler vials (Thermo Scientific, catalog number: C4011-13 )

  5. UV cuvettes (Fisher Scientific, catalog number: NC0628994 )

  6. 0.2-micron syringe filter, Nylon (Fisherbrand, catalog number: 13100108 )

  7. Poly(2-oxazoline) triblock copolymer (P[MeOx37-b-BuOx23-b-MeOx37]-piperazine) was synthesized as described previously (Luxenhofer et al., 2010)

  8. Drugs were purchased from either Adooq Bioscience, Apex Bio, or LC laboratories and stored at -20 °C

  9. Ethanol 200 proof (Fisher Scientific)

  10. Normal Saline (Teknova, catalog number: S5815 )

Equipment

  1. Pipettes (Fisher Scientific)

  2. Sonicator (Branson 2510 ultrasonic bath)

  3. Eppendorf heating block (Fisher Scientific, catalog number: 11-715-1250 )

  4. Desiccator (LabCorp)

  5. Rotor vacuum evaporator (Buchi)

  6. Vortex mixer (Fisher Scientific, catalog number: 02215365 )

  7. Benchtop microcentrifuge (Thermo Scientific)

  8. Eppendorf Centrifuge Minispin (Fisher Scientific, catalog number: 05-090-100 )

  9. HPLC (Agilent 1200 series)

  10. Zetasizer (Malvern)

Procedure


  1. Preparation of drug-loaded POx micelles (Figure 1, Small, μL scale; applicable to single and multi-drug loaded POx micelles)

    1. Prepare stock solutions of the drug and polymer in a common solvent.

      Note: Commonly used solvents for the preparation of POx micelles are ethanol (200 proof), methanol, and acetone. In some cases, a mixture of two solvents is used to dissolve drugs. The concentration of the stock solution is determined by the solubility of the drug/polymer in the solvent. E.g., Prepare Paclitaxel stock solution in ethanol at a concentration of 10 mg/ml and POx stock solution in the same solvent at a concentration of 10 mg/ml (POx is also soluble in acetone and methanol at ≥ 10mg/ml). You will need to use an ultrasonic bath for the dissolution of PTX in ethanol.

    2. Mix the polymer and drug solutions at a predetermined polymer/drug ratio (w/w).

      Note: Polymer:drug weight ratio of 10:2 is a good starting point to determine if the polymer can solubilize the drug. If the thin film disperses well in saline (step 4), work your way up to higher ratios (10:4 and 10:8). E.g., for a 10:4 loading ratio of POx:PTX, add a 50 μl stock solution of POx with a 20 μl stock solution of PTX and mix using a vortex mixer (setting 10; for ~10 s).

    3. Evaporate the solvent by placing the Eppendorf tube containing the drug-polymer mixture in a heating block and blowing a stream of nitrogen gas into the tube.

      The appearance of a clear (not cloudy) thin film is indicative of optimal heating conditions (see Figure 2A). The optimum temperature for evaporation is dictated by the physicochemical properties of the solvent (e.g., vaporization temperature) and drug (e.g., Lipophilicity, polarity, etc.) in use. Of note, the optimized temperature of evaporation for Resiquimod and PTX is 50 °C and 45 °C, respectively, and for other drugs varies from 40 °C to 80 °C.

      Note: A disposable pipette tip can be inserted into the end of a PVC tubing and lowered into the Eppendorf tube to facilitate a gentle flow of nitrogen. Further, residual traces of solvent can be removed by placing the thin film in the desiccator overnight.



      Figure 1. POx micelle preparation workflow (created with BioRender.com)


    4. Hydrate the thin film with normal saline.

      The volume of saline to be used is determined by the desired final drug(s) concentration. The optimal conditions of hydration vary with each drug. For instance, incubation for 10 min at RT followed by mild agitation by gently flicking the tube is sufficient for solubilizing thin films of Resiquimod. In comparison, Paclitaxel requires incubation at 65 °C for 20 min and gentle agitation every 5 min. The temperature of hydration should be optimized empirically for every single and multi-drug incorporated POx micelles.

      Note: Gentle agitation (using a vortex mixer or gently flicking the tube) is necessary to completely disperse the thin polymer-drug film. Visual inspection is a primary means of telling if the hydration conditions worked. While most drugs solubilize within 10 min, for certain drugs, an additional incubation time can yield better size distribution (Figure 2C) by facilitating specific interactions between the drug and polymer (hydrophobic, hydrogen bonding, etc.)

    5. Centrifuge the micelle solution at 10,000 × g for 2-3 min to separate the unencapsulated drug (pellet) and transfer the supernatant containing the drug-loaded micelles to a new tube.

    6. Depending on the stability of the POx micelle solution of drugs, these can either be stored at 4 °C for up to a couple of weeks or freeze-dried for long-term storage. The lyophilized formulation of POx micelles can be easily re-dispersed in DI water without loss in drug-loading and activity.


  2. Preparation of drug-loaded POx micelles (Figure 1, large, mL scale; applicable to single and multi-drug loaded POx micelles)

    1. Follow Steps A1 to A2. Use glass vials to accommodate large volumes of stock solutions and a round-bottom (RB) flask for mixing the drug and polymer solutions.

    2. Evaporate the solvent using a rotary vacuum evaporator. Adjust the temperature of the water bath as needed.

    3. The thin film will form in the bottom half of the RB flask when completed.

    4. Hydrate the thin film with saline and immerse it in the water bath at the optimal hydration temperature for the required time – usually 10 to 15 min.

    5. The micelle solution can be aliquoted into small volumes and centrifuged as in Step A5.

    6. Lyophilize the formulation for long-term storage.


  3. Characterization of POx micelles

    The primarily used physicochemical characterization techniques for POx micelles include dynamic light scattering (DLS) for the determination of the size distribution and reverse-phase high-performance liquid chromatography for drug loading.

    1. Sample Preparation for DLS measurement

      1. Dilute the micelles 10-fold in saline.

        Note: Typical dilutions used for DLS measurements of POx micelles range from 1:2-1:50, which correspond to drug concentrations of 0.1 mg/ml to 2 mg/ml.

      2. Use nylon syringe filters of 0.2 microns to separate large particles.

      3. Transfer the filtered solution into a clean cuvette without introducing air bubbles.

      4. Place the cuvette in the sample holder and, following equilibration for at least 2 min, take 3 separate measurements for every sample.

    2. Sample preparation for HPLC measurement

      1. Dilute the micelle sample 50-fold in a mixture of 50/50 acetonitrile (ACN): water (v/v).

      2. Filter the diluted sample using a 0.2-micron syringe filter and transfer 100 μl into an HPLC autosampler vial.

      3. Get rid of any air bubbles from the autosampler vial by gently tapping before placing it in the vial tray.

      4. Prepare the standards by serially diluting the analyte at concentrations ranging from 6.25 μg/ml to 200 μg/ml.

      5. The run settings must be optimized for each drug.

        Note: 60/40 ACN: water, 1 mg/ml flow rate, and 10 μl injection volume is usually a good starting point for most drugs. Spiking the mobile phase solvents with 0.1% trifluoroacetic acid helps sharpen the peaks. Make sure the settings used for the sample are identical to the ones used for standards.

Data analysis

  1. DLS Analysis

    The zeta sizer measures fluctuations in the scattered light intensity with time caused by the Brownian motion of nanoparticles to estimate the hydrodynamic size of nanoparticles. DLS analyzes the raw data using two algorithms, viz., cumulant analysis and distribution analysis. The cumulant analysis reports two values: Z-average, which is the mean value of the particle size distribution, and polydispersity index (PDI), which is analogous to variance. A PDI value lower than 0.2 is indicative of monodisperse particles. The cumulant analysis also provides a correlogram (correlation curve), which reports the decay rate of the signal from the sample. The amplitude of the correlogram is representative of signal to noise ratio. The distribution analysis provides Gaussian distributions of the particle size by number, volume, and intensity. Sample concentration, presence of large aggregates, and impurities or marks on the exterior of the DLS cuvettes can all influence the size distribution. More information about data interpretation can be found at https://www.malvernpanalytical.com.



    Figure 2. Representative images of the (A, D) thin film, (B, E) hydrated thin film, and (C, F) DLS volume distribution. The top panel represents drug-incorporated micelles prepared using optimal conditions, as demonstrated by the monodisperse volume distribution with a single peak around 27 nm. The bottom panel is representative of sub-optimal conditions, indicated by the cloudy thin film and the incomplete hydration of the thin film, resulting in polydisperse size distribution with a peak at about 105 nm and a large peak of aggregates in the micrometer range.

    Note: The DLS data were obtained from a separate experiment and do not represent the size distribution of the micelles shown in the images and is used here for demonstration purpose only.


  2. Drug Loading

    Chose the wavelength at which the analyte shows maximum absorbance and then integrate the peak in the chromatogram to obtain the area, which corresponds to the concentration of the drug in the sample. The retention time of the sample should compare to that of standards. Once the sample concentration is determined from the standard curve, parameters such as loading efficiency and loading capacity can be obtained from the following equations:

    Mdrug: The amount of drug incorporated in the micelle

    Mexcipient: The amount of polymer used in the formulation

    Mdrug added: The amount of drug initially fed

Acknowledgments

The original work (Vinod et al., 2020) was funded by the National Cancer Institute (NCI) Alliance for Nanotechnology in Cancer (U54CA198999, Carolina Center of Cancer Nanotechnology Excellence).

Competing interests

A.V.K. is co-inventor on patents pertinent to the subject matter of the present contribution and A.V.K. and M.S.P. have co-founders’ interest in DelAqua Pharmaceuticals Inc. having intent of commercial development of POx based drug formulations. The other authors have no competing interests to report.

References

  1. Alves, V. M., Hwang, D., Muratov, E., Sokolsky-Papkov, M., Varlamova, E., Vinod, N., Lim, C., Andrade, C. H., Tropsha, A. and Kabanov, A. (2019). Cheminformatics-driven discovery of polymeric micelle formulations for poorly soluble drugs. Sci Adv 5(6): eaav9784.
  2. Di, L., Fish, P. V. and Mano, T. (2012). Bridging solubility between drug discovery and development. Drug Discov Today 17(9-10): 486-495.
  3. Gala, U. H., Miller, D. A. and Williams, R. O., 3rd (2020). Harnessing the therapeutic potential of anticancer drugs through amorphous solid dispersions. Biochim Biophys Acta Rev Cancer 1873(1): 188319.
  4. Han, Y., He, Z., Schulz, A., Bronich, T. K., Jordan, R., Luxenhofer, R. and Kabanov, A. V. (2012). Synergistic combinations of multiple chemotherapeutic agents in high capacity poly(2-oxazoline) micelles. Mol Pharm 9(8): 2302-2313.
  5. He, Z., Wan, X., Schulz, A., Bludau, H., Dobrovolskaia, M. A., Stern, S. T., Montgomery, S. A., Yuan, H., Li, Z., Alakhova, D., Sokolsky, M., Darr, D. B., Perou, C. M., Jordan, R., Luxenhofer, R. and Kabanov, A. V. (2016). A high capacity polymeric micelle of Paclitaxel: Implication of high dose drug therapy to safety and in vivo anti-cancer activity. Biomaterials 101: 296-309.
  6. Luxenhofer, R., Schulz, A., Roques, C., Li, S., Bronich, T. K., Batrakova, E. V., Jordan, R. and Kabanov, A. V. (2010). Doubly amphiphilic poly(2-oxazoline)s as high-capacity delivery systems for hydrophobic drugs. Biomaterials 31(18): 4972-4979.
  7. Vinod, N., Hwang, D., Azam, S. H., Van Swearingen, A. E. D., Wayne, E., Fussell, S. C., Sokolsky-Papkov, M., Pecot, C. V. and Kabanov, A. V. (2020). High-capacity poly(2-oxazoline) formulation of TLR 7/8 agonist extends survival in a chemo-insensitive, metastatic model of lung adenocarcinoma. Sci Adv 6(25): eaba5542.

简介

[摘要]许多新的药物开发候选物是具有低水溶性的高度亲脂性化合物。在需要大剂量和静脉内注射的情况下,将这类化合物用于癌症治疗是一个巨大的挑战(Di等人,2012)。在这里,我们提出了一种基于聚(2-恶唑啉)聚合物(POx)的纳米配方策略,以溶解和输送疏水性药物。POx胶束通过简单的薄膜水合方法制备。在这种方法中,将药物和聚合物溶解在普通溶剂中并混合,然后在温和的加热条件下将溶剂蒸发以形成薄膜。当用盐水水合时,胶束自发形成。POx中的疏水性药物的纳米制剂是在于它具有高载药量的能力,这是优于常规的表面活性剂的胶束是唯一的。此外,可以在同一POx胶束中包含多种活性药物成分(API),从而实现二元和三元药物组合的代码传递(Han等人,2012; He等人,2016)。


[背景技术]最近的统计数据表明,只有3.7%的进入临床测试的新药候选药物被批准用于癌症治疗。这主要归因于水溶性差的候选药物的不良药代动力学,这导致了次优的性能(Gala et al。,2020)。POx聚合物胶束相对于传统的药物传递系统(例如脂质体,微粒和纳米凝胶)具有许多优势。POx的胶束对大量的各种疏水性药物无可比拟的高溶解能力使得更大量的药物具有基本上较少量的赋形剂的递送(Luxenhofer等人,2010年,他等人,2016) 。基于POx的药物制剂易于制备,安全且稳定。此外,已开发出定量结构-性质关系(QSPR)模型来预测药物向POx胶束中的负载,可用于促进高通量筛选难溶性药物开发候选物并入POx胶束中(Alves等,2019 )。

关键字:亲脂性的, 聚(2-恶唑啉), 纳米制剂, 表面活性剂, 药物活性成分, 聚合物胶束

材料和试剂
PVC管(Nalgene,¼” ID)
Eppendorf管(Fisher Scientific,目录号:05-408-129)
移液器技巧(Fisher Scientific)
11毫米塑料自动进样器样品瓶(Thermo Scientific,目录号:C4011-13)
紫外线比色皿(Fisher Scientific,目录号:NC0628994)
0.2微米注射器式过滤器,尼龙(Fisherbrand,目录号:13100108)
如先前所述(Luxenhofer et al。,2010)合成聚(2-恶唑啉)三嵌段共聚物(P [MeOx 37 -b-BuOx 23 -b-MeOx 37 ]-哌嗪)
药物购自Adooq Bioscience,Apex Bio或LC实验室,并储存在-20 °C下
乙醇200证明(Fisher Scientific)
生理盐水(Teknova,目录号:S5815)

设备


移液器(Fisher Scientific)
超声仪(Branson 2510超声浴)
Eppendorf加热块(Fisher Scientific,目录号:11-715-1250)
干燥器(LabCorp)
转子真空蒸发器(Buchi)
涡旋混合器(Fisher Scientific,目录号:02215365)
台式微量离心机(Thermo Scientific)
Eppendorf离心机Minispin(Fisher Scientific,目录号:05-090-100)
HPLC(Agilent 1200系列)
Zetasizer(马尔文)

程序


制备载药POx胶束(小,μL规模;适用于载有单药和多药的POx胶束)
在普通溶剂中准备药物和聚合物的储备溶液。
注意:制备POx胶束的常用溶剂是乙醇(200标准标准液),甲醇和丙酮。在某些情况下,可以使用两种溶剂的混合物来溶解药物。储备溶液的浓度取决于药物/聚合物在溶剂中的溶解度。例如,在乙醇中以10 mg / ml的浓度制备紫杉醇储备溶液,在同一溶剂中以10 mg / ml的浓度制备POx储备溶液(POx也以≥10mg / ml的形式溶于丙酮和甲醇中)。您将需要使用超声浴将PTX溶解在乙醇中。
以预定的聚合物/药物比率(w / w)混合聚合物和药物溶液。
注意:聚合物:药物的重量比为10:2是确定聚合物是否可以溶解药物的良好起点。如果薄膜在盐水中的分散性很好(第4步),请按照更高的比例(10:4和10:8)进行操作。例如,对于POx:PTX的负载比为10:4的情况,添加50 μ痘与20升原液μ PTX的L贮备溶液,并混合使用涡旋混合器(设定10;为〜10秒)。

通过将装有药物-聚合物混合物的Eppendorf管放在加热块中并将氮气流吹入管中,从而蒸发掉溶剂。
透明(不浑浊)的薄膜的出现表明最佳的加热条件(参见图2A)。蒸发的最佳温度取决于所用溶剂的物理化学性质(例如,蒸发温度)和药物的物理化学性质(例如,亲脂性,极性等)。值得注意的是,瑞西莫德和PTX的最佳蒸发温度分别为50 °C和45 °C ,其他药物的最佳蒸发温度为40 °C 。 °C至80 °C 。

注意:可以将一次性移液器吸头插入PVC管的末端,然后放低到Eppendorf管中,以促进氮气的缓慢流动。此外,可以通过将薄膜放在干燥器中放置过夜来去除残留的痕量溶剂。



图1. POx胶束制备工作流程(由BioRender.com创建)

请在正文中引用图1。


用生理盐水水合薄膜。
所用盐水的量取决于所需的最终药物浓度。水合的最佳条件因每种药物而异。例如,在室温下孵育10分钟,然后轻轻摇动试管轻轻搅动,足以溶解瑞西莫德的薄膜。相比之下,紫杉醇需要在65°C下孵育20分钟,每5分钟轻轻搅拌一次。对于每种结合了单药和多药的POx胶束,应根据经验优化水合温度。

注意:必须彻底搅拌(使用涡旋混合器或轻轻轻拂试管),才能完全分散聚合物药物薄膜。目视检查是判断水合条件是否有效的主要方法。虽然大多数药物会在10分钟内溶解,但对于某些药物,额外的孵育时间可通过促进药物与聚合物之间的特定相互作用(疏水,氢键等)产生更好的粒径分布(图2C)。

将胶束溶液以10,000 × g离心2-3分钟,以分离未封装的药物(小丸),并将含有载药胶束的上清液转移至新试管中。 
根据药物的POx胶束溶液的稳定性,它们可以在4°C下保存长达数周,也可以冷冻干燥以长期保存。POx胶束的冻干制剂可以轻松地重新分散在去离子水中,而不会降低药物载量和活性。

制备载药POx胶束(放大,mL刻度;适用于载有单药和多药的POx胶束)
请按照步骤A1到A2。使用玻璃瓶来容纳大量储备溶液,并使用圆底(RB)烧瓶混合药物和聚合物溶液。
使用旋转真空蒸发仪蒸发溶剂。根据需要调整水浴的温度。
完成后,薄膜将在RB烧瓶的下半部形成。
用盐水水合薄膜,并在最佳水合温度下将其浸入水浴中所需的时间-通常为10至15分钟。
可以按照步骤A5的方法将胶束溶液分装成小体积并离心。
将制剂冻干以长期保存。

POx胶束的表征
POx胶束主要使用的物理化学表征技术包括用于确定粒径分布的动态光散射(DLS)和用于载药的反相高效液相色谱法。

用于DLS测量的样品制备
在盐水中将胶束稀释10倍。
注意:用于DLS测量POx胶束的典型稀释度为1:2-1:50,对应于0.1 mg / ml至2 mg / ml的药物浓度。

使用0.2微米的尼龙注射器过滤器分离大颗粒。
将过滤后的溶液转移到干净的比色杯中,不要引入气泡。
将比色皿放在样品架中,平衡至少2分钟后,对每个样品进行3次独立测量。
用于HPLC测量的样品前处理
在50/50乙腈(ACN):水(v / v)的混合物中将胶束样品稀释50倍。
使用0.2微米的注射器过滤器和转移100过滤器的稀释样品μ升到HPLC自动进样器小瓶中。
获得通过gentl摆脱从自动进样瓶中的气泡的Ÿ将其放置在样品瓶盘前攻。
通过以6.25μg/ ml至200μg / ml的浓度范围连续稀释分析物来制备标准液。
必须针对每种药物优化运行设置。
注意:60/40 ACN:水,1毫克/毫升的流速,和10 μ升注射体积是通常对大多数药物的良好起点。用0.1%三氟乙酸加标流动相溶剂有助于锐化色谱峰。确保用于样品的设置与用于标准的设置相同。


数据分析


DLS分析
zeta粒度仪可测量由纳米粒子的布朗运动引起的散射光强度随时间的波动,以估算纳米粒子的流体动力学尺寸。DLS使用两种算法(即累积量分析和分布分析)分析原始数据。累积量分析报告两个值:Z平均值(是粒度分布的平均值)和多分散指数(PDI),类似于方差。PDI值低于0.2表示单分散颗粒。累积量分析还提供了相关图(相关曲线),该相关图报告了来自样品的信号的衰减率。相关图的幅度代表信噪比。分布分析按数量,体积和强度提供了粒度的高斯分布。样品浓度,大聚集体的存在以及DLS比色杯外部的杂质或痕迹都会影响尺寸分布。有关数据解释的更多信息,请访问https://www.malvernpanalytical.com。



图2.(A,D)薄膜,(B,E)水合薄膜和(C,F)DLS体积分布的代表性图像。上图代表使用最佳条件制备的掺入药物的胶束,如单分散体积分布所示,在27 nm附近有一个峰。底部面板代表次优条件,其由浑浊的薄膜和薄膜的不完全水合指示,导致多分散的粒度分布,其峰值在约105 nm处,聚集体的峰值在微米范围内。

注意:DLS数据是从单独的实验中获得的,并不代表图像中所示胶束的尺寸分布,此处仅用于演示目的。


载药量
选择分析物显示最大吸光度的波长,然后对色谱图中的峰进行积分以获得面积,该面积与样品中药物的浓度相对应。样品的保留时间应与标准样品的保留时间进行比较。一旦从标准曲线确定了样品浓度,就可以从以下方程式获取诸如上样效率和上样能力等参数:


液相色谱

LE


M drug :胶束中掺入的药物量

M赋形剂:配方中聚合物的用量

M药物添加量:最初投放的药物量


致谢


原始工作(Vinod等,2020)由美国国家癌症研究所(NCI)癌症纳米技术联盟(U54CA198999,卡罗来纳州癌症纳米技术卓越中心)资助。


利益争夺


AVK是与本发明主题相关的专利的共同发明人,而AVK和MSP在DelAqua Pharmaceuticals Inc.的共同创始人中感兴趣,其目的是基于POx的药物制剂的商业开发。其他作者没有竞争利益要报告。


参考


Alves,VM,Hwang,D.,Muratov,E.,Sokolsky-Papkov,M.,Varlamova,E.,Vinod,N.,Lim,C.,Andrade,CH,Tropsha,A.和Kabanov,A.( 2019)。化学信息学驱动的难溶性药物聚合胶束制剂的发现。Sci Adv 5(6):eaav9784。
Di,L.,Fish,PV and Mano,T.(2012年)。在药物发现和开发之间架起桥梁。今日毒品网(Drug Discov)17(9-10):486-495。
UH的晚会,DA的Miller和RO的Williams,第3版(2020年)。通过无定形固体分散体利用抗癌药的治疗潜力。Biochim Biophys Acta Rev Cancer 1873(1):188319。
Han,Y.,He,Z.,Schulz,A.,Bronich,TK,Jordan,R.,Luxenhofer,R.和Kabanov,AV(2012)。高容量聚(2-恶唑啉)胶束中多种化学治疗剂的协同组合。Mol Pharm 9(8):2302-2313。
他Z.,Wan.X.,Schulz,A.,Bludau,H.,Dobrovolskaia,MA,Stern,ST,Montgomery,SA,Yuan,H.,Li,Z.,Alakhova,D.,Sokolsky,M 。,Darr,DB,Perou,CM,Jordan,R.,Luxenhofer,R. and Kabanov,AV(2016)。紫杉醇的高容量聚合物胶束:高剂量药物治疗对安全性和体内抗癌活性的影响。生物材料101:296-309。
Luxenhofer,R.,Schulz,A.,Roques,C.,Li,S.,Bronich,TK,Batrakova,EV,Jordan,R.和Kabanov,AV(2010)。双亲两性聚(2-恶唑啉)作为疏水性药物的高容量传递系统。生物材料31(18):4972-4979。
Vinod,N.,Hwang,D.,Azam,SH,Van Swearingen,AED,Wayne,E.,Fussell,SC,Sokolsky-Papkov,M.,Pecot,CV和Kabanov,AV(2020)。TLR 7/8激动剂的高容量聚(2-恶唑啉)制剂可延长在化学敏感性低的肺腺癌转移模型中的生存期。Sci Adv 6(25):eaba5542。
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引用:Vinod, N., Hwang, D., Azam, S. H., Van Swearingen, A. E. D., Wayne, E., Fussell, S. C., Sokolsky-Papkov, M., Pecot, C. V. and Kabanov, A. V. (2021). Preparation and Characterization of Poly(2-oxazoline) Micelles for the Solubilization and Delivery of Water Insoluble Drugs. Bio-protocol 11(6): e3959. DOI: 10.21769/BioProtoc.3959.
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