Controlling Morphology and Release Behavior of Sorafenib-Loaded

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Materials and Interfaces

Controlling Morphology and Release Behavior of Sorafenibloaded Nanocarriers prepared by Flash Nanoprecipitation Mingwei Wang, Shan Lin, Junyou Wang, Lei Liu, Wenjuan Zhou, Rizwan Bhutto Ahmed, Aiguo Hu, Xuhong Guo, and Martien Abraham Cohen Stuart Ind. Eng. Chem. Res., Just Accepted Manuscript • DOI: 10.1021/acs.iecr.8b02105 • Publication Date (Web): 16 Aug 2018 Downloaded from http://pubs.acs.org on August 22, 2018

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Industrial & Engineering Chemistry Research

Controlling Morphology and Release Behavior of Sorafenib-loaded Nanocarriers prepared by Flash Nanoprecipitation Mingwei Wang†‡, Shan Lin†‡, Junyou Wang*†, Lei Liu†, Wenjuan Zhou†, Rizwan Bhutto Ahmed†, Aiguo Hu⊥, Xuhong Guo*†§, and Martien A. Cohen Stuart*† †

State Key Laboratory of Chemical Engineering, School of Chemical Engineering, East China

University of Science and Technology, Shanghai 200237, P. R. China. ⊥

School of Materials Science and Engineering, East China University of Science and

Technology, Shanghai 200237, P. R. China. §

Engineering Research Center of Materials Chemical Engineering of Xinjiang Bingtuan, Shihezi

University, Xinjiang 832000, P. R. China E-mail: [email protected]; [email protected]; [email protected]

KEYWORDS. Flash nanoprecipitation, drug-carrier NPs, controlled morphology, release behavior

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ABSTRACT.

Flash Nanoprecipitation (FNP) is a recent developed method featuring fast processing and simple equipment for preparing drug-carrier NPs. Herein, we prepared stable sorafenib-loaded NPs with biocompatible amphiphilic poly(ethylene glycol)-block-poly(lactide acid) (PEG-b-PLA) as stabilizing polymer based on FNP. The formed NPs show well-controlled size and high drug loading content compared with nanoparticles from traditional anti-solvent precipitation. Moreover, drug/polymer mass ratio (D/P) and stream velocity presented as Reynolds number (Re) show strong effects on particles size and internal morphology. Low D/P ratio and Re number provide core-shell nanoparticles with drug nuclei distributed in PLA matrix, which could release the sorafenib completely but keep the polymer aggregates after the drug release. While high D/P ratio and Re number lead to grained nanoparticles with bigger size and low packing density due to the co-precipitation of the PEG blocks in the structure. The drug release of these particles is fast and typically accompanied with the dissociation of the nanoparticles. Our study demonstrates that the particle internal morphology and solute packing density are crucial factors to manipulate the drug release of the FNP nanoparticles, and the developed strategy could be widely adopted to assess drug release of FNP nanoparticles for further therapeutic applications.

1. Introduction When properly designed, polymeric nanoparticles (NPs) with controlled structure and properties can be promising drug-carriers: the protecting polymers can minimize drug degradation, and also achieve targeted delivery and increased bioavailability of the drug.1-3 In recent years, considerable efforts have been devoted to developing novel formulation approaches and techniques for preparing drug-carrier NPs.4-14 Among the different achievements, flash


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nanoprecipitation (FNP) established by Johnson and Prud’homme, combines advantages of fast processing, simple equipment, and good control of particle size and size distribution.15-17 FNP relies on rapid mixing of a solvent stream containing the (usually) hydrophobic solute plus the stabilizing copolymer, with an anti-solvent, in a confined impinging jet (CIJ) mixer or a multiinlet vortex mixer (MIVM).17-19 The fast mixing, on the order of milliseconds, creates a uniform local supersaturation which helps to separate the phases of nucleation and growth in the process. The co-precipitation typically involves amphiphilic molecules as nuclei stabilizer, and leads to nanoparticles with controlled size and fairly narrow size distribution.20 In contrast to selfassembly approaches in which hydrophobic solutes are encapsulated through aggregation of amphiphilic molecules, and which lead to thermodynamic equilibrium nanoparticles, FNP normally produces kinetically frozen structures with high drug loading,20, 21 and also offers more control over particles size and morphology, by tuning experimental parameters like solvent/antisolvent ratio, flow rate, solute concentration and type of protecting copolymer, and as such it is a versatile technique for dispersing functional ingredients.22-27 Despite these many advantages, not all compounds can be dispersed by means of FNP. In particular, low LogP solutes remain a challenge.28 Because low solubility in water is essential for high supersaturation and fast nucleation, FNP works best for very hydrophobic solutes. Indeed, Zhu et al have found that solute with LogP above 12 (calculated by ACD model) could form very stable nanoparticles, and there size and distribution hardly change over time.29 Prud’homme et al demonstrate bifenthrin with LogP ~ 7.3 could form stable nanoparticles.30 Empirically, LogP >6 is proposed as the required hydrophobicity in order to form stable nanoparticles.28 Solutes of lower LogP do not readily form stable nanoparticles with stabilizer polymers, although ternary combinations, with extra co-precipitated hydrophobic molecules, can also form


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stable nanoparticles.28,

31, 32

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Apparently, the co-precipitation of very hydrophobic molecules

enhances the nucleation rate, which is crucial for obtaining sufficiently stable nanoparticles. Unfortunately, the functional ingredient in such particles ends up in compact solid cores within the particles, and this slows down their release rate, and even hampers their complete release.24, 33, 34

For example, PEG-b-PLGA-based nanoparticles loaded with paclitaxel (PTX)-silicate

prodrugs could only release about 50% of drugs, and the release tended to be slower for prodrugs having greater hydrophobicity.33 York et. al found that, generally, molecules in FNP nanoparticles are released much less easily than those in self-assembled particles.24 All these findings demonstrate that the drug release behavior of nanoparticles appears to be a crucial issue in the context of application

Scheme 1. a: Chemical structures of amphiphilic copolymer PEG-b-PLA and sorafenib; b:illustration of flash nanoprecipitation preparing NPs with different morphology.


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of FNP nanoparticles. Nevertheless, for nanoparticles made by FNP, the relationship between solute release and particle preparation protocol has not been investigated yet. In the current work, we investigate a system that allows to prepare FNP nanoparticles with controlled internal morphology and release of solutes. We select a cancer drug with low LogP (5.1, sorafenib) as our hydrophobic solute, and a biocompatible amphiphilic block copolymer poly(ethylene glycol)-b-poly(lactic acid) (PEG-b-PLA, 5k-b-10k) as the stabilization polymer. Using the MIVM, stable nanoparticles are obtained, even without co-stabilizer or co-precipitated hydrophobic compound. Upon increasing the drug/polymer mass ratio and the flow rate, the particle size increases and, more noticeably, the structure changes from a simple core-shell morphology of drug nuclei dispersed in the PLA matrix, to a grained morphology implying coprecipitation of hydrophilic PEG blocks. Both the core-shell and grained particles can completely release the drug, but with different fate of the polymer: the core-shell particle leaves and empty polymer particle behind, while the grained one is completely disintegrated after the drug release. Our study demonstrates that drug release depends crucially on particle morphology, and shows that the latter can be manipulated. Therapeutic applications may therefore benefit from the strategy adopted here. 2. Results and Discussion The chemical structures of the drug and stabilized copolymer are shown in Scheme 1. Sorafenib with a LogP around 5.1 (ACD model) is an anti-cancer drug, applied widely for treatment of liver and kidney cancers.35-37 The amphiphilic stabilizer, PEG-b-PLA is a biocompatible and popular copolymer for preparing drug-carrier NPs. To control mixing rate, we select the multi-inlet vortex mixer in this study; a view of its top cross-section is shown in


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Scheme 1. For preparing NPs, both PEG-b-PLA and sorafenib are dissolved in the watermiscible organic solvent tetrahydrofuran (THF, stream 1); the other three streams (2 - 4) are pure water. For comparison, we run a control experiment in which the NPs are made by traditional anti-solvent precipitation. Polymer and sorafenib concentrations are fixed at 1mg/ml and 0.25 mg/ml, respectively, for both methods. The THF solution either enters the MIVM, with THF and water flow velocities of 12 and 36 ml/min, respectively, or is simply pipetted into the water phase at the same THF/H2O volume ratio, under stirring with a magnetic stir bar. As shown in Figure 1a, nanoparticles from FNP have a hydrodynamic radius (Rh) around 74 nm and a narrow size distribution, whereas particles from traditional precipitation show bigger sizes and larger spread in size. For the FNP nanoparticles, the drug-loading content (DLC) and encapsulation efficiency, calculated from UV absorption, and are found to be 7.6% and 31%, respectively, (Table S1) while these values for the traditional precipitation are respectively 2.6% and 11%, which is about three times lower than those for FNP

Figure 1. a: Size and size distribution of NPs prepared by FNP and self-assembly method at same polymer and drug concentration; b: drug loading content and drug loading efficiency of NPs prepared by FNP and traditional precipitation method.


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nanoparticles. It turns out that, indeed, FNP is more favorable for preparing smaller nanoparticles with high drug loading. In the following, we assess the influence of experimental factors controlling FNP preparation such as drug/polymer mass ratio and flash flow velocity. 2.1 Influence of drug to polymer mass ratio First, the effect of drug to polymer mass ratio (D/P) is investigated; we do this at a constant polymer concentration and variable sorafenib concentrations. The polymer concentration is fixed at 1 mg/mL in order to be sure of that sofafenib could still be soluble completely in THF even at high D/P ratio. The inlet flow velocities of THF and water are kept at 12 and 36 ml/min, respectively. As shown in Figure 2a, the pure diblock copolymer without any sorafenib forms nanoparticles with a hydrodynamic radius (Rh) around 60 nm, as obtained from dynamic light scattering. Introducing sorafenib leads to bigger NPs; the size keeps increasing up to 174 nm with increasing D/P up to the maximum ratio of 10/1. Figure 2b suggests that the NPs prepared at low D/P ratio have a low PDI about 0.12, indicating a narrow distribution of the nanoparticles. Both the size and the size distribution (PDI) increase with increasing D/P ratio and at D/P ratio of 5/1 aggregates appear with sizes up to 1 µm. (Figure S1, Table S2) Nanoparticles at 0.25/1, 1/1 and 5/1 are selected representatively and the size change at these three D/P ratios, at 4 oC are recorded as a function of time, over 15 days. Figure 2c indicates that the average particle sizes at D/P of 0.25/1 and 1/1 hardly change, while those for 5/1 decrease from 152 nm to 117 nm. This is likely due to sedimentation of the big aggregates, which is consistent with size distribution results from Figure 2b. In order to confirm this hypothesis, we have agitated the particle solution after 15 days and measured again with light scattering, and found that the light scattering


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intensity and sizes increase markedly due to redispersion of the big aggregates, (Table S3) whereas the samples at D/P of 0.25/1 show similar intensity and size before and after the agitation. Clearly, the nanoparticles should be prepared preferably at low D/P ratio in order to control the stability and dispersity.

Figure 2. (a) Hydrodynamic radius (Rh) of NPs prepared at different drug/polymer mass ratios (D/P), the blue arrows indicate the selected D/P nanoparticles for size distribution and stability test; (b) polydispersity index (PDI) of NPs prepared at different D/P; (c) the stability of NPs at different D/P ratio over time; (d) ratio of gyration radius over hydrodynamic radius, Rg/Rh as a function of D/P. The polymer concentration was fixed at 1mg/mL, and the velocities of THF and water streams were 12 and 36 ml/min.


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The FNP nanoparticle are further characterized by multi-angle static light scattering. Such angular dependent measurements provide the gyration radius (Rg); once this is known, the ratio of gyration over hydrodynamic radius (Rg/Rh), can be determined, which is a measure of the packing density of the micellar core.38, 39 It is proposed that hard spheres display a Rg/Rh ratio around 0.78, while polymer coil in good solvent show a Rg/Rh ratio ~ 1.5 to 1.7. In our work, we

Figure 3. TEM images of NPs prepared by drug/polymer mass ratio, D/P of 0.25/1 (a) and 5/1 (b). Drug release of NPs prepared at D/P ratio of 0.25/1 and 5/1 monitored by light scattering (c) and UV-Vis absorbance at wavelength of 267 nm (d).


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find that Rg/Rh increases from 1.0 to 1.5 (Figure S2), indicating an apparent decrease of the core packing density upon increasing D/P from 0.25/1 to 10/1. To further confirm this density decrease, we have made TEM images of nanoparticles prepared at D/P ratios of 0.25/1 and 5/1. Figure 3a shows that the nanoparticles at D/P ratio of 0.25/1 have core-shell morphology, featuring densely packed spherical cores with a diameter around 140 nm. In contrast, nanoparticles at 5/1 (Figure 3b) have a diameter around 310 nm and a ‘grained’ core structure. Taking the modest length of the diblock copolymer into account, we can exclude the possibility that all diblocks have their PLA block inside and their PEG block at the surface; hence, some PEG must have co-precipitated with the drug in the core of the particles to form the inhomogeneous, grained structure visible in the TEM images. Probably, the hydrophilic PEO blocks create small water-rich domains inside the particles, alongside more drug enriched domains; this could explain the grained structures. The differences in morphology are likely to have consequences for the release rate of sorafenib: the Kelvin effect implies that solubility of particles increases as the inverse of the particle radius: the very small domains visible in Figure 3b will probably dissolve much more easily than the much larger ones in Figure 3a.40, 41 We therefore checked whether morphology indeed influences the drug release. Nanoparticles at D/P ratio of 0.25/1 and 5/1 were again selected, and the drug release at room temperature were monitored by both light scattering and UV-vis absorbance. In Figure 3(c, d), the decay of scattering intensity over time monitors the presence of the nanoparticles, while the UV absorption shows the concentration of non-released sorafenib. For particles at low D/P, the light scattering intensity decreases to a lower limit of about 40%, while the UV-vis data suggests an almost complete release of sorafenib after about 24 hours. The difference between the light scattering and UV results suggests that there are still particles in solution despite complete


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release of drug; most likely these are some sort of polymer micelles. We propose that the low drug concentration creates a limited number of nuclei which are taken up in a continuous PLA matrix, which stabilizes the organic assembly. As a result, even after the complete release of the sorafenib a hydrophobic core is left which renders the particle stability. Nanoparticles at 5/1, in contrast, show a fast release of the drug, and this release process is accompanied by disappearance of the nanoparticles. Most likely, the particles disintegrate into very small entities, but these are not stable and form loose aggregates that settle. To confirm the proposed mechanism, we agitate the solution after 24 hours, and find that both the light scattering intensity and radius increases a lot due to the redispersion of these aggregates. A similar test performed on the 0.25/1 sample shows very little changes in intensity and size (Table S4).

From UV

absorption, we determine as the drug concentration encapsulated in the nanoparticles about 0.0097 mg/ml, for both 0.25/1 and 5/1, corresponding to a drug loading ratio around 7.1 % (Table S1). That the drug loading ratio from core-shell and grained NPs is rather similar implies that their different release rates must be assigned to the differences in primary grain size, as expressed by the Kelvin effect. Hence, the release data is consistent with light scattering and TEM findings. 2.2 Influence of Reynolds number In this section, we consider the influence of the mixing intensity, in terms of the Reynolds number (Re) calculated based on the stream velocity (Table S5).20, 42 We fix the D/P ratio at 1/1 and the organic stream velocity at 12 ml/min, and vary aqueous stream velocity systematically from 12 to 96 ml/min. As shown in Figure 4a, upon increasing Re from 700 to 2200 the particle get bigger: the hydrodynamic radius increases from 75 nm to 150 nm. (Figure 4a) Nanoparticles at low Re particles show broad size distributions and, consequently, lower stability over time due


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to the sedimentation of bigger aggregates. Increasing Re number leads to NPs with narrower size distribution and improved stability as indicated by the constant size within 15 days (Figure 4b, 4c, and Figure S3). Moreover, Figure 4d suggests that Rg/Rh ratio of NPs increases with increasing Re number, which implies that the packing density of the particles decreases with increasing Re

Figure 4. (a) Hydrodynamic radius (Rh) of NPs prepared at different mixing velocity presented as Reynolds number (Re), the blue arrows indicate the selected Re nanoparticles for size distribution and stability test; (b) PDI of NPs prepared at different Re; (c) the stability of NPs at different Re over time; (d) Rg/Rh controlled by Re. The drug and polymer concentration was constant at 1mg/mL in THF solution, and drug/polymer mass ratio is 1: 1.


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number. (Figure S4) These trends are reminiscent to that observed upon increasing D/P. It therefore seems likely that the NPs at low Re number form core-shell morphology, while particles at high Re number, with bigger size and lower density, have again more grained structures. This is confirmed by TEM images (Figure 5a, 5b) where we see core-shell particles at Re number of 700, and inhomogeneous, grained structures for particles at Re number of 2200. The release kinetics are investigated as well, and once again, we find that for nanoparticles with core-shell morphology and high packing density, prepared at low Re, polymer aggregates continue

Figure 5. TEM images of NPs prepared at Re number of 700 (a) and 2200 (b). Drug release of NPs prepared at Re number of 700 and 2200 by light scattering (c) and UV-Vis absorbance at wavelength of 267 nm (d).


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to exist in solution after the sorafenib release, but nanoparticles of low packing density and grained morphology, prepared at high Re, show both release of sorafenib and dissociation of the particles over time. (Figure 5c, 5d) Combined with the findings from the D/P study, we conclude that FNP nanoparticles in the current study show increased size, decreased packing density and different morphologies with increasing D/P ratio or Re number, which results in significant differences in release behavior.

2.3 Multiple influence and proposed mechanism In order to further confirm that size, internal morphology and packing density can be tuned by D/P and Re, we explored more combinations emphasizing extreme conditions, that is low D/P ratio at low Re number, or high D/P ratio at high Reynolds number. Figure 6a, for D/P ratio of 0.25/1, shows that increase of Re indeed induces an increase of Rh from 63 nm to 73 nm. Nanoparticles at the high D/P ratio 5/1 shows a similar increase of the particle size upon increasing Re number. On the other hand, at fixed Re, increased D/P ratio also leads to increased size of nanoparticles. As can be seen in Figure 6b, the nanoparticle at 0.25-700, and 5-2200 indeed show the lowest and highest Rg/Rh ratios, respectively, confirming again that the core density decreases with either increasing D/P ratio or Re number. Since the particle size hardly influence the drug release, the internal morphology and packing density must be the key parameters that underlie the differences in release behavior of the nanoparticles. Low D/P ratio and Re number lead to core-shell nanoparticle with monolithic sorafenib cores embedded in PLA. In this case, the somewhat slower mixing and/or lower drug


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Figure 6. Hydrodynamic radius (a) and Rg/Rh (b) of NPs prepared under different combination of D/P ratio and Re number.

concentration lead to a lower nucleation rate and a lower concentration of nuclei; it then follows that these nuclei can grow to larger sizes. These particles can release sorafenib completely, but keep the polymer aggregated in solution. In contrast, at high D/P ratio and high Re, the nucleation rate is so high that many tiny nuclei are formed which consume nearly all available drug. Hence, the nuclei hardly grow, but associate with polymer to unstable primary particles which subsequently form spherical mixed aggregates, eventually stabilized by left-over diblock copolymer. This scenario leads to bigger nanoparticles with a structure composed of both PEG domains and tiny sorafenib particles, resulting in low packing density and grained appearance of the nanoparticles. The small drug nuclei play a role in keeping the particle together; when they dissolve fast, the particles fall apart leaving behind very small organic particles which tend to form loose aggregates. To our best knowledge, this is the first report demonstrating this variation


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of structure and packing density, with impact on the release behavior of nanoparticles prepared based on FNP. Moreover, we have tried three other solutes with different LogP in order to investigate the general applicability of the control on structure and release. We find that nanoparticles from β-carotene (LogP 15.5) and vitamin E succinate (LogP 11.8) do not change that much on the Rg/Rh ratio upon changing Re number (Figure S5), indicating limited variations on the packing density and internal structure. Both the nanoparticles with β-carotene and vitamin E succinate at low and high Re number hardly release the solutes over time (Figure S6). Different with these high LogP solutes, gefitinib with a LogP around 4.1 show similar changes on Rg/Rh ratio and release behavior with that of sorafenib nanoparticle at low and high Re number. These findings suggest that the internal structure of a particle with a solute having a moderately low LogP would be easier to be controlled than the one with a high LogP.

3. Conclusion Sorafenib has been encapsulated in nanoparticles by means of solvent/anti-solvent precipitation by both conventional mixing and Flash Nano Precipitation (FNP). FNP displays clear advantages in terms of controlling the particle size and increasing drug loading content. The obtained nanoparticles are stable over time. Remarkable changes in size and internal morphology occur in FNP upon increasing drug/polymer (D/P) ratio and flash flow velocity represented as Reynolds number Re). In particular, at low D/P ratio and Re number one obtains core-shell nanoparticles with monolithic drug cores embedded in the PLA matrix, which can release the sorafenib completely, leaving behind ‘empty’ polymer micellar aggregates. In contrast, at high D/P ratio and Re one gets lead bigger nanoparticles with low packing density and a grained internal morphology, indicating co-precipitation of the PEG blocks in the structure. The drug release from these particles is fast and typically accompanied by dissociation of the nanoparticles. All


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these tunable structures and properties can be manipulated by control parameters of the multiinlet vortex mixer, which is virtually impossible by traditional precipitation approaches. Moreover, our study demonstrates that the internal morphology and packing density of nanoparticles are key parameters controlling the drug release of FNP nanoparticles. The insights obtained here will hopefully benefit attempts to improve understanding of drug release of FNP nanoparticles containing more hydrophobic solutes.

4. Experiment Materials. PEG-b-PLA (5k-b-10k) was purchased from Jinan Daigang Biomaterial Co, Ltd. Sorafenib was purchased from Guangzhou Isun Pharmaceutical Co., Ltd. Tetrahydrofuran (THF) in chemically pure grade was purchased from Shanghai Tianlian Fine Chemical Co., Ltd. Pure water was obtained by a Milli-Q water purification system and was used in all experiments. Other reagents and solvents were used as received without any further treatment. Characterization. Nanoparticle morphology was observed on a JEOL JEM-1400 TEM instrument with an acceleration voltage of 100 kV. One drop of the nanoparticle solution was deposited on carbon-coated copper grid. The droplet was allowed to dry under ambient conditions. Light sacttering was performed at 25 oC with an ALV-CGS3 light scattering apparatus, operating at a wavelength of 632.8 nm. The mean hydrodynamic radius (Rh) of NPs were measureed at fixed angle of 90o, while the gyration radius (Rg) of NPs were measured from the angular dependence of scattering (Guinier plot), varying the angle from 40o to 150o. The CONTIN method is used to analyze the distribution of particle radii. For data processing, average and standard deviations were obtained from six duplicates with each acquisition times of


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20 s. UV-vis absorption spectra of samples were recorded on a UV-1600 UV-vis spectrophotometer. Preparation of NPs. The NPs were prepared by the FNP through a MIVM system. Sorafenib was added into a solution of PEG-b-PLA (5000-b-10000 g/mol) in THF. The organic solution was fed (stream 1), along with water (streams 2 - 4), into a MIVM (Scheme 1) system using two digitally controlled syringe pumps (Harvard Apparatus, PHD 2000) to yield NPs. NP suspensions were subjected to ultracentrifugation (Amicon®Ultra-15 centrifugal filter, 10 K device, Millipore) with a 40o fixed angle centrifuge rotor at 6,500 g for 5 min at room temperature. The ultrafiltration process was repeated with fresh aqueous replacement at least four times to remove the organic solvent down to a barely detectable level. For preparing NPs samples with different D/P ratios, the polymer concentration was fixed at 1 mg/mL while the sorafenib concentration was varied from 0 mg/mL to 10 mg/mL, and the inlet flow velocities of THF and water are kept at 12 and 36 ml/min, respectively. For preparing NPs samples with different Re, both the polymer and drug concentration was fixed at 1 mg/mL. The stream velocity of THF was fixed at 12 ml/min and the velocity of water stream was changed from 12 to 96 ml/min systematically. Determination of DLC and DLE, and the in vitro drug release study. To determine the DLC and DLE of the nanoparticles, a calibration curve of sorafenib was first constructed based on UV-vis absorbance at 267 nm as a function of sorafenib concentration with a UV-1600 UV-vis spectrophotometer. (Figure S7 and eqs S1). Then, DLC and DLE of drug-loaded NPs were calculated as percentage according to the following eqs 1 and 2, respectively:

DLC ሺwt%ሻ =

amount of drug in NPs amount of drug loaded NPs

×100

(1)


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DLE ሺwt%ሻ =

amount of drug in NPs total amount of feeding drug

×100

(2)

The release rates of sorafenib from the NPs were investigated as following description. Briefly, a dialysis tube (Spectra/Por® Float-A-Lyzer® G2, molecular weight cut-off (MWCO): 8 – 10 kD, Spectrum Laboratories, Inc.) containing 5 mL drug-loaded NPs was immersed in 125 mL pure water at 37 oC. At designed time point, 1 mL of drug-loaded NPs solution was taken out and was characterized by ALV-CGS3 light scattering apparatus and UV-vis spectrophotometer at 37 oC. The remaining light scattering intensity of NPs solutions were measured after different time intervals using equation (3).

Remaining Intensity ሺ%ሻ=

A1 A2

×100

(3)

A1 is the light intensity at time t and A2 is the intensity at beginning 0 hour. The remaining UV absorbance were measured at 267 nm after different time intervals using equation (4).

Remaining Absorbance ሺ%ሻ=

B1 B2

×100

(4)

B1 is the absorbance at time t and B2 is the absorbance at beginning 0 hour.

ASSOCIATED CONTENT Supporting Information. The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.iecr.XXXXXX.


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Drug loading content and efficiency of NPs, light scattering intensity and size change of NPs after 15 days storage upon agitation, size and size distribution of different NPs, gyration radius of NPs, light scattering intensity and size change of NPs after 24 hour release upon agitation, Reynolds number of each run for different NPs, Rg/Rh ratio and release results of nanoparticles prepared based on β-carotene, vitamin E succinate and gefitinib at different Re number, calibration curve of sorafenib.

AUTHOR INFORMATION Corresponding Author *[email protected] *[email protected] * [email protected] Author Contributions ‡

M. W. and S. L. contributed equally.

ACKNOWLEDGMENT This work was supported by National Natural Science Foundation of China (NSFC) for Young Scholars (21706074), Shanghai Gaofeng Gaoyuan Project (SG1501A001), and Fundamental Research Funds for the Central Universities (222201814007).


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REFERENCES (1) Blanco, E.; Shen, H.; Ferrari, M., Principles of nanoparticle design for overcoming biological barriers to drug delivery. Nat. Biotechnol. 2015, 33(9), 941-951. (2) Ramasamy, T.; Ruttala, H. B.; Gupta, B.; Poudel, B. K.; Choi, H.; Yong, C. S.; Kim, J. O., Smart chemistry-based nanosized drug delivery systems for systemic applications: A comprehensive review. J. Control. Release 2017, 258, 226-253. (3) Allen, T. M.; Cullis, P. R., Drug Delivery Systems: Entering the Mainstream. Science 2004, 303(5665), 1818-1822. (4) Wu, M.; Yang, Y., Metal-Organic Framework (MOF)-Based Drug/Cargo Delivery and Cancer Therapy. Adv. Mater. 2017, 29(23), 1606134. (5) Li, B.; Meng, Z.; Li, Q.; Huang, X.; Kang, Z.; Dong, H.; Chen, J.; Sun, J.; Dong, Y.; Li, J.; Jia, X.; Sessler, J. L.; Meng, Q.; Li, C., A pH responsive complexation-based drug delivery system for oxaliplatin. Chem. Sci. 2017, 8(6), 4458-4464. (6) Mitra, S.; Sasmal, H. S.; Kundu, T.; Kandambeth, S.; Math, K.; Diaz Diaz, D.; Banerjee, R., Targeted Drug Delivery in Covalent Organic Nanosheets (CONs) via Sequential Postsynthetic Modification. J. Am. Chem. Soc. 2017, 139(12), 4513-4520. (7) Chen, W.; Ouyang, J.; Liu, H.; Chen, M.; Zeng, K.; Sheng, J.; Liu, Z.; Han, Y.; Wang, L.; Li, J.; Deng, L.; Liu, Y.; Guo, S., Black Phosphorus Nanosheet-Based Drug Delivery System for Synergistic Photodynamic/Photothermal/Chemotherapy of Cancer. Adv. Mater. 2017, 29(5), 1603864..


21


Page 22 of 27

(8) Yao, X.; Niu, X.; Ma, K.; Huang, P.; Grothe, J.; Kaskel, S.; Zhu, Y., Graphene Quantum Dots-Capped Magnetic Mesoporous Silica Nanoparticles as a Multifunctional Platform for Controlled Drug Delivery, Magnetic Hyperthermia, and Photothermal Therapy. Small 2017, 13(2). (9) Liu, Q.; Song, L.; Chen, S.; Gao, J.; Zhao, P.; Du, J., A superparamagnetic polymersome with extremely high T-2 relaxivity for MRI and cancer-targeted drug delivery. Biomaterials 2017, 114, 23-33. (10) Feng, T.; Ai, X.; An, G.; Yang, P.; Zhao, Y., Charge-Convertible Carbon Dots for Imaging Guided Drug Delivery with Enhanced in Vivo Cancer Therapeutic Efficiency. ACS Nano 2016, 10(4), 4410-4420. (11) Yahia-Ammar, A.; Sierra, D.; Merola, F.; Hildebrandt, N.; Le Guevel, X., Self-Assembled Gold Nanoclusters for Bright Fluorescence Imaging and Enhanced Drug Delivery. ACS Nano 2016, 10(2), 2591-2599. (12) Li, D.; Bu, Y.; Zhang, L.; Wang, X.; Yang, Y.; Zhuang, Y.; Yang, F.; Shen, H.; Wu, D., Facile Construction of pH- and Redox-Responsive Micelles from a Biodegradable Poly(beta-hydroxyl amine) for Drug Delivery. Biomacromolecules 2016, 17(1), 291-300. (13) Liu, B.; Chen, Y.; Li, C.; He, F.; Hou, Z.; Huang, S.; Zhu, H.; Chen, X.; Lin, J., Poly(Acrylic Acid) Modification of Nd3+-Sensitized Upconversion Nanophosphors for Highly Efficient UCL Imaging and pH-Responsive Drug Delivery. Adv. Funct. Mater. 2015, 25(29), 4717-4729.


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Page 23 of 27 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


(14) Fang, Q.; Wang, J.; Gu, S.; Kaspar, R. B.; Zhuang, Z.; Zheng, J.; Guo, H.; Qiu, S.; Yan, Y., 3D Porous Crystalline Polyimide Covalent Organic Frameworks for Drug Delivery. J. Am. Chem. Soc. 2015, 137(26), 8352-8355. (15) Johnson, B. K.; Prud'Homme, R. K., Mechanism for Rapid Self-Assembly of Block Copolymer Nanoparticles. Phys. Rev. Lett. 2003, 91(11), 118301-118302. (16) Johnson, B. K.; Prud'Homme, R. K., Flash NanoPrecipitation of organic actives and block copolymers using a confined impinging jets mixer. Aust. J. Chem. 2003, 56(10), 1021-1024. (17) Johnson, B. K.; Prud'Homme, R. K., Chemical processing and micromixing in confined impinging jets. AIChE J. 2003, 49(9), 2264-2282. (18) Liu, Y.; Cheng, C.; Liu, Y.; Prud'Homme, R. K.; Fox, R. O., Mixing in a multi-inlet vortex mixer (MIVM) for flash nano-precipitation. Chem. Eng. Sci. 2008, 63(11), 2829-2842. (19) Akbulut, M.; Ginart, P.; Gindy, M. E.; Theriault, C.; Chin, K. H.; Soboyejo, W.; Prud'Homme, R. K., Generic method of preparing multifunctional fluorescent nanoparticles using flash NanoPrecipitation. Adv. Funct. Mater. 2009, 19(5), 718-725. (20) Zhu, Z., Effects of amphiphilic diblock copolymer on drug nanoparticle formation and stability. Biomaterials 2013, 34(38), 10238-10248. (21) Zhu, Z.; Anacker, J. L.; Ji, S.; Hoye, T. R.; Macosko, C. W.; Prud'Homme, R. K., Formation of Block Copolymer-Protected Nanoparticles via Reactive Impingement Mixing. Langmuir 2007, 23(21), 10499-10504.


23


Page 24 of 27

(22) Zhu, Z.; Margulis-Goshen, K.; Magdassi, S.; Talmon, Y.; Macosko, C. W., Polyelectrolyte stabilized drug nanoparticles via flash nanoprecipitation: a model study with β-carotene. J. Pharm. Sci.-US 2010, 99(10), 4295-4306. (23) Saad, W. S.; Prud'Homme, R. K., Principles of nanoparticle formation by flash nanoprecipitation. Nano Today 2016, 11(2), 212-227. (24) York, A. W.; Zablocki, K. R.; Lewis, D. R.; Gu, L.; Uhrich, K. E.; Prud'Homme, R. K.; Moghe, P. V., Kinetically Assembled Nanoparticles of Bioactive Macromolecules Exhibit Enhanced Stability and Cell-Targeted Biological Efficacy. Adv. Mater. 2012, 24(6), 733739. (25) Liu, Y.; Kathan, K.; Saad, W.; Prud'Homme, R. K., Ostwald Ripening of β-Carotene Nanoparticles. Phys. Rev. Lett. 2007, 98(3), 036102. (26) Wang, M.; Xu, Y.; Wang, J.; Liu, M.; Yuan, Z.; Chen, K.; Li, L.; Prud'Homme, R. K.; Guo, X., Biocompatible Nanoparticle Based on Dextran-b-Poly(L-Lactide) Block Copolymer Formed by Flash Nanoprecipitation. Chem. Lett. 2015, 44(12), 1688-1690. (27) Wang, M.; Yang, N.; Guo, Z.; Gu, K.; Shao, A.; Zhu, W.; Xu, Y.; Wang, J.; Prud'Homme, R. K.; Guo, X., Facile Preparation of AIE-Active Fluorescent Nanoparticles through Flash Nanoprecipitation. Ind. Eng. Chem. Res. 2015, 54(17), 4683-4688. (28) Tam, Y. T.; To, K.; Chow, A., Fabrication of doxorubicin nanoparticles by controlled antisolvent precipitation for enhanced intracellular delivery. Colloid. Surface. B. 2016, 139, 249-258.


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Page 25 of 27 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


(29) Zhu, Z. X., Flash Nanoprecipitation: Prediction and Enhancement of Particle Stability via Drug Structure. Mol. Pharmaceut. 2014, 11(3), 776-786. (30) Liu, Y.; Tong, Z.; Prud'Homme, R. K., Stabilized polymeric nanoparticles for controlled and efficient release of bifenthrin. Pest Manag. Sci. 2008, 64(8), 808-812. (31) Margulis, K.; Magdassi, S.; Lee, H. S.; Macosko, C. W., Formation of curcumin nanoparticles by flash nanoprecipitation from emulsions. J. Colloid Interf. Sci. 2014, 434, 65-70. (32) Chow, S. F.; Wan, K. Y.; Cheng, K. K.; Wong, K. W.; Sun, C. C.; Baum, L.; Chow, A., Development of highly stabilized curcumin nanoparticles by flash nanoprecipitation and lyophilization. Eur. J. Pharm. Biopharm. 2015, 94, 436-449. (33) Han, J.; Michel, A. R.; Lee, H. S.; Kalscheuer, S.; Wohl, A.; Hoye, T. R.; McCormick, A. V.; Panyam, J.; Macosko, C. W., Nanoparticles Containing High Loads of Paclitaxel-Silicate Prodrugs: Formulation, Drug Release, and Anticancer Efficacy. Mol. Pharmaceut. 2015, 12(12), 4329-4335. (34) Shi, L.; Shan, J. N.; Ju, Y. G.; Aikens, P.; Prud'Homme, R. K., Nanoparticles as delivery vehicles for sunscreen agents. Colloid. Surface. A. 2012, 396, 122-129. (35) Liu, Y.; Feng, L.; Liu, T.; Zhang, L.; Yao, Y.; Yu, D.; Wang, L.; Zhang, N., Multifunctional pH-sensitive polymeric nanoparticles for theranostics evaluated experimentally in cancer. Nanoscale 2014, 6(6), 3231-3242.


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Page 26 of 27

(36) Wang, H.; Wang, H.; Yang, W.; Yu, M.; Sun, S.; Xie, B., Improved Oral Bioavailability and Liver Targeting of Sorafenib Solid Lipid Nanoparticles in Rats. Aaps Pharmscitech 2018, 19(2), 761-768. (37) Lin, T.; Gao, D.; Liu, Y.; Sung, Y.; Wan, D.; Liu, J.; Chiang, T.; Wang, L.; Chen, Y., Development and characterization of sorafenib-loaded PLGA nanoparticles for the systemic treatment of liver fibrosis. J. Control. Release 2016, 221, 62-70. (38) Guan, Z.; Cotts, P. M.; McCord, E. F.; McLain, S. J., Chain Walking: A New Strategy to Control Polymer Topology. Science 1999, 283(5410), 2059. (39) Kunz, D.; Thurn, A.; Burchard, W., Dynamic light scattering from spherical particles. Colloid Polym. Sci. 1983, 261(8), 635-644. (40) Dolenc, A.; Govedarica, B.; Dreu, R.; Kocbek, P.; Srčič, S.; Kristl, J., Nanosized particles of orlistat with enhanced in vitro dissolution rate and lipase inhibition. Int. J. Pharmaceut. 2010, 396(1), 149-155. (41) Skinner, L. M.; Sambles, J. R., The Kelvin equation—a review. J. Aerosol Sci. 1972, 3(3), 199-210. (42) Lince, F.; Bolognesi, S.; Stella, B.; Marchisio, D. L.; Dosio, F., Preparation of polymer nanoparticles loaded with doxorubicin for controlled drug delivery. Chem. Eng. Res. Des. 2011, 89(11), 2410-2419.


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Controlling Morphology and Release Behavior of Sorafenib-Loaded

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