Drug loading augmentation in polymeric nanoparticles using
a coaxial turbulent jet mixer: Yong investigator perspective
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Lim, Jong-Min et al. “Drug loading augmentation in polymeric
nanoparticles using a coaxial turbulent jet mixer: Yong investigator
perspective.” Journal of Colloid and Interface Science 538 (2019):
45-50 © 2019 The Author(s)
As Published
10.1016/J.JCIS.2018.11.029
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Elsevier BV
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Author's final manuscript
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https://hdl.handle.net/1721.1/125309
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Drug Loading Augmentation in Polymeric Nanoparticles Using a
Coaxial Turbulent Jet Mixer: Yong Investigator Perspective
Jong-Min Lim1,*, Truong Cai2, Stefan Mandaric3, Sunandini Chopra3, Hyeonwoo Han1,
Seokkyu Jang1, Won Il Choi4, Robert Langer2,5, Omid C. Farokhzad6,7, and Rohit Karnik3
1Department of Chemical Engineering, Soonchunhyang University, Asan, Chungnam, 31538,
Republic of Korea
2Department of Chemical Engineering, Massachusetts Institute of Technology, Cambridge, MA
02139, United States
3Department of Mechanical Engineering, Massachusetts Institute of Technology, Cambridge, MA
02139, United States
4Center for Convergence Bioceramic Materials, Convergence R&D Division, Korea Institute of
Ceramic Engineering and Technology, 202, Osongsaengmyeong 1-ro, Osong-eup, Heungdeok- gu, Cheongju, Chungbuk 28160, Republic of Korea
5David H. Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology,
Cambridge, MA 02139, United States
6Laboratory of Nanomedicine and Biomaterials, Department of Anesthesiology, Brigham and
Women’s Hospital, Harvard Medical School, Boston, MA 02115, United States
7King Abdulaziz University, Jeddah 22254, Saudi Arabia
Abstract
Hypothesis—In conventional ‘bulk’ nanoprecipitation, the capacity to load hydrophobic drugs into the polymeric nanoparticles (NPs) is limited to about 1%. The size distribution of the resulting NPs becomes polydisperse when higher precursor concentration is used to increase the drug loading. Hence, it should be possible to enhance the hydrophobic drug loading in polymeric NPs while maintaining the uniform NP size distribution by optimizing the nanoprecipitation process and purification process.
*Corresponding Authors jmlim@sch.ac.kr. Telephone: 82-41-530-4961. (J.-M. Lim). Notes
In compliance with the Brigham and Women’s Hospital and Harvard Medical School institutional guidelines, O.C.F. discloses his financial interest in BIND Therapeutics, Selecta Biosciences, and Blend Therapeutics, three biotechnology companies developing NP technologies for medical applications. BIND, Selecta and Blend did not support the aforementioned research, and currently these companies have no rights to any technology or intellectual property developed as part of this research. In compliance with MIT institutional guidelines, R.L. discloses his financial interest in BIND Therapeutics, Selecta Biosciences, Blend Therapeutics, and Kala, four biotechnology companies developing NP technologies for medical applications. BIND, Selecta, Blend, and Kala did not support the aforementioned research, and currently these companies have no rights to any technology or intellectual property developed as part of this research.
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Author manuscript
J Colloid Interface Sci
. Author manuscript; available in PMC 2020 March 07.Published in final edited form as:
J Colloid Interface Sci. 2019 March 07; 538: 45–50. doi:10.1016/j.jcis.2018.11.029.
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Experiments—Systematical studies were performed to enhance the loading of docetaxel (Dtxl) by using a process of centrifugal spin-down, rapid mixing by turbulence, and addition of co-solvent. The size distributions and Dtxl loading of the NPs were measured using dynamic light scattering and HPLC, respectively.
Findings—The centrifugal spin-down process helps to maintain uniform size distribution even at the high precursor concentration. In the bulk nanoprecipitation, the resulting NPs achieved Dtxl loading up to 3.2%. By adopting turbulence for rapid mixing, the loading of Dtxl increased to 4.4%. By adding hexane as co-solvent, the loading of Dtxl further increased to 5.5%. Because of the drug loading augmentation, high degree of control, and extremely high production rate, the developed method may be useful for industrial-scale production of personalized nanomedicines by nanoprecipitation.
Graphical Abstract
Keywords
Nanoparticles; nanomedicines; nanoprecipitation; rapid mixing; drug loading
1. Introduction
Various therapeutic agents that are capable of inhibiting the metabolism pathways of diseased cells and tissues have been developed during the past decades [1–4]. Due to the intrinsic toxicity of these agents, however, they are limited to a certain dose to maintain the overall beneficial effect. Targeted nanoparticles (NPs) have the capabilities to overcome the dose-limiting factor by delivering a much larger fraction of the medicine to tumor tissue area. Therefore, the development of targeted NP platforms can indeed begin a new age of well-designed and tunable release of drug that would revolutionize the field of pharmacy [5– 12].
Docetaxel (Dtxl) is a semisynthetic taxane approved for treatment of several major solid tumor cancers, including breast, prostate, lung, and, gastric [13]. Polyethylene glycol (PEG) and poly (lactic-co-glycolic acid) (PLGA) are FDA approved biocompatible and
biodegradable polymer, which is widely used as a model platform for controlled drug delivery carriers [14–16]. Recently, Dtxl-loaded targeted poly (lactic-co-glycolic acid)-polyethylene glycol (PLGA-PEG) polymeric NPs reached Phase II clinical trials for prostate cancer therapy [11, 17]. A large range of formulation parameters and NP physicochemical properties need to be evaluated pre-clinically during the development of such polymeric NPs. Although nanoprecipitation methods are widely adopted to prepare polymeric NPs in laboratory scale due to its simplicity and versatility, the Dtxl drug loading that can maintain uniform NP size distribution is limited [18]. While most of the other parameters such as size, PEG coverage, surface charge, and targeting ligand density can be controlled within their
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desirable range of values relatively easily in conventional nanoprecipitation methods [19], enhancing the drug loading has been more difficult because large hydrophobic drug
aggregates are formed during the conventional nanoprecipitation process [17, 18]. Therefore, other methods such as emulsion solvent evaporation are often favored, despite the difficulty in achieving small NP sizes that are optimal for drug delivery [20–22].
Here we demonstrated simple and versatile methods to enhance the loading of hydrophobic drug in polymeric NPs synthesized by nanoprecipitation. To enhance the Dtxl loading, we systematically studied the following three modifications of NP preparation process. First, we could remove Dtxl aggregates, which were originated from the high initial loading of Dtxl, by an additional centrifugation step prior to NP washing. Second, we could enhance the drug loading as well as explore the possibilities of scaling up with better control over the
nanoprecipitation process by using a coaxial jet turbulent mixer [23]. Finally, we enhanced the drug loading further with the addition of hexane as co-solvent that would trap the Dtxl and increase the chance to be encapsulated in the hydrophobic PLGA core of PLGA-PEG NPs. The novel strategy has strong possibility to reduce the gap between NP formulation prepared in academic laboratories and that in pharmaceutical industry thanks to enhanced hydrophobic drug loading in polymeric NPs and extremely high production rate.
2. Experimental section
2.1. Preparation of PLGA-PEG Nanoparticles.
NPs were prepared by conventional nanoprecipitation method [18] and rapid
nanoprecipitation using coaxial turbulent jet mixer [23]. Briefly, PLGA27K-PEG5K block
copolymer (Boehringer Ingelheim GmbH) and docetaxel (Dtxl, LC laboratories) were dissolved in acetonitrile (ACN, Sigma-Aldrich). To enhance the Dtxl loading, small amount of organic co-solvent (i.e., hexane) was added into the organic precursor solution. Deionized water was used as anti-solvent during both conventional ‘bulk’ nanoprecipitation and rapid nanoprecipitation. In the case of conventional nanoprecipitation, the polymer precursor solution was added drop-wise into deionized water at different ratios (i.e., R = 0.1 or 0.05) over about 2 h under magnetic stirring. For rapid nanoprecipitation, coaxial turbulent jet mixer was prepared as described elsewhere [23] by inserting 23 G blunt needle (337 μm I.D. and 641.4 μm O.D., Strategic applications Inc.) into Tee union tube fittings made of PTFE (Plasmatech Co.) and fixing via optical adhesive (NOA81, Norland products) under UV light. PTFE tubing (Plasmatech Co.) with inner diameters D = 3.175 mm was connected to the tee union tube fitting. The polymer precursor solution and deionized water were used as inner and outer stream, respectively. To generate and maintain turbulence (Re = 2056), the flow rates of inner and outer stream were controlled by syringe pumps (Harvard Apparatus). The viscosity, μ, and density, ρ, of water-ACN mixtures were used for the calculation of the Reynolds number, Re [24].
2.2. Purification and Washing of PLGA-PEG Nanoparticles.
In conventional washing by ultrafiltration, as synthesized PLGA-PEG NP suspensions were purified by ultrafiltration at 4000 rpm for 5 min using Amicon Ultracel 100 K membrane filters, washed twice with water, and resuspended in water or PBS. In spin-down process to
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remove Dtxl aggregates, an aliquot of the as synthesized PLGA-PEG NP solution was spun down by centrifugation at 3000 rpm for 1 min and supernatant was collected using
micropipette. The collected supernatant was subsequently purified by the ultrafiltration at 4000 rpm for 5 min using Amicon Ultracel 100 K membrane filters.
2.3. Characterization of Nanoparticles.
The size distributions by volume percent of the synthesized PLGA-PEG NPs were measured using dynamic light scattering (Zetasizer Nano ZS, Malvern Instruments Ltd). Three measurements were performed on each sample, and the distribution is obtained as an average of the three measurements along with standard deviation. The synthesized PLGA-PEG NPs were stained by uranyl acetate (Electron Microscopy Sciences) and imaged using TEM (JEOL 200CX). Docetaxel loading in the PLGA-PEG NPs was measured by HPLC (Agilent Technologies, 1100 Series) using established procedures [18].
3. Results and Discussions
3.1. Drug Loading Augmentation in Bulk Synthesis Methods.
Conventional bulk nanoprecipitation method is widely adopted to prepare NPs from amphiphilic block copolymer in academic research laboratories because simplicity and versatility. As shown in Scheme 1a, drug loaded NPs can be prepared simply by adding an organic precursor solution into the aqueous anti-solvent under magnetic stirring [18]. Recently, we demonstrated rapid nanoprecipitation method to make drug loaded NPs using a coaxial turbulent jet mixer, which has extremely high NP productivity (Scheme 1b) [23]. In both cases, the NP size distribution becomes polydisperse when the initial loading of hydrophobic drug (e.g., Dtxl) is increased, which mainly originated from the precipitation of hydrophobic drug itself [18]. In previous studies, the concentration of the polymeric
precursor and initial loading of the hydrophobic drug (i.e., the nominal ratio of drug to polymer by weight) were limited to avoid large hydrophobic drug aggregates and the synthesized drug loaded NPs were typically washed by ultrafiltration (Scheme 1c) [18]. However, we found that ultrafiltration does not effectively separate the drug aggregates from the NPs, and the aggregates tend to grow in size with time. Here, we hypothesized that these large aggregates can be separated from NPs by centrifugation, leaving behind NPs with higher drug loading. We therefore introduced an additional spin-down process, which consisted of a short centrifugation step to settle down most of large hydrophobic drug aggregates and a collection step to recover supernatant with NPs from the sample (Scheme 1d).
In our drug loading optimization experiments, Dtxl loaded PLGA27k-PEG5k NPs (Scheme
1e) were used because they are widely used as a model system [13, 15] and adopted in clinical trials [8, 11, 17]. When 10 mg/mL PLGA27k-PEG5k precursor and 5 % initial
loading of Dtxl was used for conventional bulk nanoprecipitation, the size distribution of as synthesized resulting NPs was uniform (Figure 1a). Because there were no Dtxl aggregates in the solution, the size distribution of resulting NPs did not change significantly after the spin-down process (Figure 1b). After removal of unencapsulated Dtxl using ultrafiltration, the final loading of Dtxl in PLGA27k-PEG5k NPs was measured by HPLC. As shown in
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Figure 1c, the loading amount of Dtxl was not altered by the spin-down process, which confirms that there were no Dtxl aggregates in the suspension under these conditions of nanoprecipitation. When the PLGA27k-PEG5k precursor concentration was increased to 50
mg/mL and the initial drug loading of Dtxl was increased to 10% in conventional bulk nanoprecipitation, the size distribution of as synthesized resulting NPs was not uniform, as evidenced by the size distribution curve as well as its irreproducibility that results in large error bars (Figure 1d). However, the size distribution of the NPs became uniform after the spin-down process, which is consistent with our hypothesis that the spin-down process settles down the Dtxl aggregates (Figure 1e). As shown in Figure 1f, the amount of Dtxl aggregates in the solution (i.e., 4.9 % in drug loading) can be estimated from the Dtxl amount of as synthesized NPs (i.e., 8.1% in drug loading) minus the Dtxl amount after spin-down process (i.e., 3.2% in drug loading).
We summarized the Dtxl loading in PLGA27k-PEG5k NPs after the spin-down process from
various precursor concentrations and initial Dtxl loadings in Figure 2. The ratio of polymer precursor solution to deionized water (i.e., R) was controlled in conventional bulk
nanoprecipitation process (Figure 2a and 2b). We could categorize the process into three different regions based on the uniformity of NP size distribution before and after the spin-down process. When 10 mg/mL PLGA27k-PEG5k precursor and 5 to 10 % initial loading
was used, NPs are uniform in their size distribution both before and after the spin-down process, as indicated by the blue solid line in Figure 1. However, the drug loading for this case is low, about 1.3 % for R = 0.1. In the cases of 30 to 50 mg/mL PLGA27k-PEG5k
precursor and 5 to 10 % initial loading as indicated by the green dashed line, the NPs are uniform only after the spin-down process. In these conditions the drug loading could be enhanced to 3.2 % after spin-down process for R = 0.1. When the initial loading is increased to 20 %, the NPs are not uniform regardless of the spin- down process and the PLGA27k
-PEG5k concentration as indicated by the red dashed dotted line. Since the Dtxl aggregates
cannot be removed completely even after the spin-down process as confirmed by the polydispersity in size distribution, the drug loading data in the red dashed dotted region are not reliable. As a result, we concluded that the maximal drug loading after spin down process can be achievable in 50 mg/mL of PLGA27k-PEG5k precursor with 10 % initial
loading of Dtxl, while maintaining the uniform size distribution.
3.2. Drug Loading Augmentation Using Coaxial Turbulent Jet Mixer.
The coaxial turbulent jet mixer was used to examine the effect of rapid mixing on the drug loading enhancement, because it can synthesize uniform polymeric NPs by rapid mixing in a high-throughput manner (i.e., up to 3 kg/d) [23]. We used 50 mg/mL of PLGA27k-PEG5k
with 10 % initial loading of Dtxl for the synthesis of NPs by the coaxial turbulent jet mixer, because Dtxl loading in PLGA27k-PEG5k NPs could be maximized for this condition while
keeping the uniformity of the size distribution after the spin-down process by the bulk nanoprecipitation method as discussed earlier. The as synthesized Dtxl loaded PLGA27k
-PEG5k NPs from the coaxial turbulent jet mixer had a smaller average size and more
uniform size distribution than that from the bulk synthesis method (Figure 1d and Figure 3a). However, the dynamic light scattering data appeared slightly unstable when the data from multiple measurements were averaged (Figure 3a). After the spin-down process, the
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Dtxl loaded PLGA27k-PEG5k NPs became more stable and uniform (Figure 3b). With the
turbulent jet mixer, the difference in Dtxl loading in PLGA27k-PEG5k NPs with and without
the spin-down process was not statistically significant (Figure 3c). It is noteworthy that the Dtxl loading in PLGA27k-PEG5k NPs was 4.4 % when the coaxial turbulent jet mixer was
used, which is higher by a factor 1.40, simply by changing the synthesis method from the bulk nanoprecipitation to the rapid mixing by turbulence (Figure 4a). Therefore, rapid mixing reduced the amount of Dtxl aggregates and increased loading. In the case of rapid mixing by turbulence, the growth of Dtxl aggregates is limited due to the extremely fast mixing time (< 10 ms). As a result, the Dtxl seed can be more easily covered by the PLGA- PEG block copolymer, which increases the chance for the Dtxl molecules to be encapsulated inside the PLGA-PEG NPs.
3.3. Drug Loading Augmentation by adding co-solvent in Coaxial Turbulent Jet Mixer.
We further explored the enhancement of drug loading by hypothesizing that the addition of organic co-solvent can promote the encapsulation of Dtxl in the PLGA-PEG NPs. When the rapid mixing by turbulence occurs, the small amount of organic co-solvent traps the Dtxl and increases the partitioning of the drug molecules in the PLGA part of the PLGA-PEG NPs. To perform the specific role, the organic co-solvent should be immiscible with water while being miscible with PLGA, Dtxl, and ACN. In this condition, the solvent will co-precipitate to form emulsion NPs. Hexane is an ideal candidate for the study because it is only slightly soluble in water (i.e., 9.5 mg/L), while it can dissolve Dtxl and PLGA27k (experimentally
measured solubility of 0.8 and 0.2 g/L, respectively). In addition, hexane has low toxicity. In case of rapid mixing by turbulence, the addition of small amount of hexane increased the drug loading in NPs while maintaining the uniformity of the size distribution after the spin-down process (Figure 4a). At a hexane concentration of 30 mg/mL, however, the drug loading was reduced (Figure 4a) and the size distribution was not uniform regardless the spin-down process (Figure 4b and 4c). As the concentration of hexane increased, all of hexane might not be covered by PLGA-PEG at some point, which is 30 mg/mL in aforementioned system. The non-uniformity in size distribution may originate from the formation of Dtxl aggregates in excess hexane, which was not covered by the PLGA-PEG block copolymer. While maintaining the narrow size distribution, the loading of Dtxl in PLGA-PEG NPs could be increased about 4.2 times compared to that in conventional bulk synthesis methods by combining the additional spin down process, the rapid mixing by turbulence, and the additional organic co-solvent. In addition, the coaxial turbulent jet mixer could continuously synthesize PLGA-PEG NPs suitable to industrial-scale production (i.e., up to 3 kg/day).
4. Conclusion
We demonstrated the enhancement of hydrophobic anticancer drug loading in polymeric NPs by optimizing the purification process and adopting the coaxial turbulent jet mixer. In conventional bulk synthesis method with high concentration of Dtxl or PLGA-PEG in precursor solution, the as synthesized NP are not uniform in size mainly due to the
hydrophobic drug aggregates [18]. As a result, the hydrophobic drug loading in synthesized PLGA-PEG NPs is limited to about 1.3% while maintaining the narrow size distribution,
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which is relevant to the previously reported values [17, 18]. The Dtxl aggregates could be removed simply by adding the centrifugal spin-down process prior to the conventional washing by ultrafiltration, which enabled to increase the hydrophobic drug loading to about 3.2% while maintaining the narrow size distribution. The drug loading could be further enhanced to 4.4% by adopting rapid nanoprecipitation using the coaxial turbulent jet mixer. While maintaining the narrow size distribution, we further enhanced the drug loading to 5.5% by adding small amount of hexane as co-solvent, which would trap the Dtxl and make it easier to be encapsulated in the hydrophobic PLGA core of PLGA-PEG NPs in water rich environment. Overall, the drug loading could be enhanced about 4.2 times compared to the conventional nanoprecipitation method [17, 18] by adopting turbulence for rapid mixing, adding organic co-solvent, and optimizing the purification process. This method enables a high degree of control and reproducibility due to rapid, continuous nanoprecipitation in the turbulent jet mixer [23], while simultaneously enhancing the drug loading. Because of the drug loading augmentation, controllability, and extremely high production rate, the development and industrial-scale production of personalized nanomedicines can be accelerated.
ACKNOWLEDGMENT
This research was supported by the Koch-Prostate Cancer Foundation Award in Nanotherapeutics (R.L. and O.C.F.), by the Concept Development Grant 5P50CA090381–09 from the Dana Farber Cancer Institute Prostate SPORE (O.C.F.), by NIH Grants EB015419 (O.C.F. and R.K.), CA119349 (R.L. and O.C.F.), and EB003647 (O.C.F.), by the National Cancer Institute Center of Cancer Nanotechnology Excellence at MIT-Harvard U54-CA151884 (R.L. and O.C.F.), and by the National Research Foundation of Korea (NRF) grant No.
NRF-2017R1C1B5018050 (J.-M.L.) and NRF-2018R1D1A1B07043620 (W.I.C.) funded by the Korea government (MSIT).
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Figure 1.
NP synthesis by conventional bulk synthesis methods. (a) Size distribution of as synthesized Dtxl loaded PLGA27k-PEG5k NPs, (b) size distribution of Dtxl loaded PLGA27k-PEG5k NPs
after the spin down process, and (c) drug loading and encapsulation efficiency of Dtxl loaded PLGA27k-PEG5k NPs prepared from 10 mg/mL PLGA27k-PEG5k precursor with 5 %
Dtxl initial loading by conventional bulk synthesis method (R = 0.1). (d) Size distribution of as synthesized Dtxl loaded PLGA27k-PEG5k NPs, (e) size distribution of Dtxl loaded
PLGA27k-PEG5k NPs after the spin down process, and (f) drug loading and encapsulation
efficiency of Dtxl loaded PLGA27k-PEG5k NPs prepared from 50 mg/mL PLGA27k-PEG5k
precursor with 10 % Dtxl initial loading by conventional bulk synthesis method (R = 0.1).
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Figure 2.
Drug loading in PLGA27K-PEG5K NPs after the spin down process. Dtxl loading in
PLGA27K-PEG5K NPs prepared by conventional bulk synthesis method with (a) R = 0.1 and
(b) R = 0.05, respectively. PLGA27K-PEG5K NPs is synthesized with various Dtxl initial
loading and PLGA27K-PEG5K precursor concentration. In the blue solid line region,
PLGA27K-PEG5K NPs are uniform in their size distribution both as synthesized and after the
spin down process. In the green dashed line region, PLGA27K-PEG5K NPs are uniform in
their size distribution only after the spin down process. In the red dashed dotted line region, PLGA27K-PEG5K NPs are not uniform in their size distribution both as synthesized and after
the spin down process.
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Figure 3.
NP synthesis using coaxial turbulent jet mixer. (a) Size distribution of as synthesized Dtxl loaded PLGA27k-PEG5k NPs, (b) size distribution of Dtxl loaded PLG27k-PEG5k NPs after
the spin down process, and (c) drug loading and encapsulation efficiency of Dtxl loaded PLGA27k-PEG5k NPs prepared from 50 mg/mL PLGA827k-PEG5k precursor with 10 % Dtxl
initial loading by using coaxial turbulent jet mixer (R = 0.1).
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Figure 4.
NP synthesis with addition of hexane as a co-solvent using coaxial turbulent jet mixer. (a) Effect of hexane in organic precursor on drug loading and average NP diameter of Dtxl loaded PLGA27k-PEG5k NPs prepared from 50 mg/mL PLGA27k-PEG5k precursor with
10 % Dtxl initial loading (R = 0.1). Here, the drug loading and the NP diameter were measured after the spin down process. Size distribution of as synthesized Dtxl loaded PLGA27k-PEG5k NPs with 30 mg/mL hexane (b) before and (c) after the spin down process.
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Scheme 1.
Schematic illustration of NP synthesis and purification methods. Schematic illustration of (a) conventional nanoprecipitation by bulk synthesis method, (b) rapid nanoprecipitation using a coaxial turbulent jet mixer, (c) conventional washing by ultrafiltration, and (d) spin down by centrifugation for removal of Dtxl precipitate. (d) TEM image of Dtxl loaded PLGA-PEG NPs prepared using the coaxial turbulent jet mixer.
