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Amphiphilic Nanoparticle-in-Nanoparticle Drug Delivery Systems Exhibiting Crosslinked Inorganic Rate-Controlling Domains Julia Talal, Inbal Abutbul-Ionita, Inbar Schlachet, Dganit Danino, and Alejandro Sosnik Chem. Mater., Just Accepted Manuscript • DOI: 10.1021/acs.chemmater.6b04922 • Publication Date (Web): 22 Dec 2016 Downloaded from http://pubs.acs.org on December 26, 2016

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Chemistry of Materials

Amphiphilic Nanoparticle-in-Nanoparticle Drug Delivery Systems Exhibiting Crosslinked Inorganic Rate-Controlling Domains Julia Talal1, Inbal Abutbul-Ionita2, Inbar Schlachet1, Dganit Danino1,3 and Alejandro Sosnik1,3* 1

Laboratory of Pharmaceutical Nanomaterials Science, Department of Materials Science and Engineering, Technion-Israel Institute of Technology, 3200003 Haifa, Israel 2 Faculty of Biotechnology and Food Engineering, Technion-Israel Institute of Technology, 3200003 Haifa, Israel 3 Russell Berrie Nanotechnology Institute (RBNI), Technion-Israel Institute of Technology, Technion City, 3200003 Haifa, Israel

*Corresponding author: Prof. Alejandro Sosnik, Department of Materials Science and Engineering, De-Jur Building, Office 607, Technion-Israel Institute of Technology, 3200003 Haifa, Israel Email: [email protected]; [email protected]

1 ACS Paragon Plus Environment


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Abstract Aiming to explore the potential of sol-gel chemistry to physically stabilize polymeric micelles and confer sustained release features, this work reports for the first time on the production of hybrid organic-inorganic multimicellar nanomaterials that, as opposed to the state-of-the-art, display crosslinked poly(siloxane) rate-controlling domains. To achieve this goal, poly(ethylene oxide)-b-poly(propylene oxide) amphiphiles of different architecture (linear and branched) and hydrophilic-lipophilic balance were primarily modified with alkoxysilane moieties through the reaction of the terminal hydroxyl groups of the copolymer and 3-(triethoxysilyl)propyl isocyanate. Then, ethoxysilane-modified polymeric micelles were prepared in water where hydrolysis resulted in a silanol-decorated surface that was cured by spray-drying. Due to the singular spraying mechanism of the Nano Spray-Dryer B-90 used in this work, that is based on a vibrating mesh spray with holes in the 4-7 µm size range that produce ultra-fine droplets, a novel kind of hybrid amphiphilic nanoparticle-in-nanoparticle system with high physical stability was developed. Comprehensive microscopy studies demonstrated the multimicellar nature of these novel nanomaterials. Moreover, they hosted high payloads of the hydrophobic model drug antiretroviral tipranavir in the hydrophobic domains, and sustained the release with a more controlled zero-order fashion than the pristine non-crosslinked counterparts that followed the classical biphasic release with an initial burst effect and a more moderate rate later on.


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Graphical abstract

Hydrolysis Drug encapsulation

Spray-drying + condensation



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1. Introduction Polymeric micelles (PMs) are colloidal materials formed by the auto-aggregation of amphiphilic copolymers above the denominated critical micellar concentration (CMC) and they emerged as one of the most auspicious nanotechnology strategies for the encapsulation of hydrophobic diagnostic and therapeutic molecules1,2. Their potential relies on the great molecular flexibility to tailor the molecular architecture and weight, the hydrophilicity/hydrophobicity balance, the size, the shape and the surface charge and hence, to optimize the encapsulation capacity and confer special features such as active cell targeting and mucoadhesiveness3,4. The European Technology Platform for Nanomedicine (ETPN) ranked PMs as the second most prominent platform for the therapy of cancer following the liposomes5. Significant advantages of PMs with respect to liposomes are greater chemical stability of the components, easier production and storage, and usually lower cost. Moreover, PMs can be processed under more extreme temperature conditions (e.g., freeze-drying, spray-drying) and withstand better the harsh conditions of the gastrointestinal tract. However, standard PMs display two striking pitfalls, namely physical instability under profuse dilution in the biological milieu6 and, when the interaction between drug and core is only of physical nature, poor ability to sustain the release7. The former drawback could be overcome by covalent crosslinking of the corona or the core, approaches that are often based on toxic bifunctional coupling agents (e.g., glutaraldehyde) or uncontrolled polymerization reactions (e.g., free radical polymerization of unsaturated precursors) using initiators or catalysts that jeopardize the molecular integrity of the encapsulated active molecule6,8,9. In this framework, the stabilization of the PM is usually carried out before the encapsulation stage and thus, its capacity to host the cargo is substantially reduced. For instance, it is not intriguing that many of the works dealing with the stabilization of PMs turn a blind eye to the investigation of the encapsulation stage8,10. Few works investigating selective crosslinking pathways have been reported to date11,12. Conversely, the limited control of the release kinetics has been addressed by the conjugation or complexation of the drug to the micellar core employing chemical links that undergo disruption under specific biological conditions (e.g., pH decrease in tumors)13,14. This strategy requires the availability of reactive functional groups (e.g., hydroxyl) that, upon hydrolysis, release an intact drug molecule; changes in the chemical structure might alter the pharmacological activity and the toxicity and preclude the use of the drug-polymer conjugate. In addition, despite the approach of


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choice, it is worth pointing out that every synthetic step compromises the scalability of the process and increases the final cost of the product. Last but not least, as most nanomedicines, drug-loaded PMs require a final drying step to preserve them until use that ensures efficient redispersion and the regeneration of structures with a similar size and size distribution of the originally produced. Owing to good biocompatibility, commercial availability and approval by regulatory agencies as pharmaceuticals and medical devices, linear (Pluronic®, poloxamers) and X-shaped (Tetronic®, poloxamines) thermo-responsive poly(ethylene oxide)-b-poly(propylene oxide) copolymers (PEO-PPOs) represent one of the most extensively investigated families of micelle- and gelforming biomaterials14,15. Over the last decade, we have comprehensively characterized the performance of pristine and chemically-modified versions of these PMs for the delivery of hydrophobic drugs by different parenteral and mucosal administration routes16–18 and demonstrated that even if they consistently improve the drug pharmacokinetics with respect to the free counterpart, they exhibit limited capacity to sustain the release in vivo. In fact, pharmacokinetic profiles usually combine a burst phase accompanied by a fast clearance within a few hours17. To expand the versatility of PMs, our research is currently devoted to the design of more complex self-assembly architectures that are stabilizable by means of milder and more selective chemistries19. The sol–gel process that results in the formation of crosslinked inorganic Si-O-Si networks is based on two consecutive reactions, the hydrolysis of an alkyl or alkoxysilane precursor to form silanol groups, followed by their condensation into siloxanes20,21. Usually, the first step takes place in a water-rich environment, while the second demands the elimination of water and is conducted under water-deprived conditions. This synthetic pathway is very selective and enables the encapsulation of eclectic payloads that vary from small molecules to living systems (e.g., bacteria), going through a broad spectrum of sensitive proteins and genes20,22–24. In addition, it is fully compatible with almost every imaginable drug because owing to their low chemical stability, silanes and silanols are not used in the design of pharmacologically active molecules. In a seminal work that introduced a new family of injectable biomedical thermo-responsive gels that crosslink under physiological conditions, we demonstrated that the condensation of silanol



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groups could also occur in water-rich medium, though that the reaction rate was extremely slow25. Aiming to explore the potential of this chemistry to physically stabilize the micelles and confer sustained release features to PMs, this work reports for the first time on the production of hybrid organic-inorganic multimicellar systems that, as opposed to the state-of-the-art, display crosslinked inorganic (poly(siloxane)) rate-controlling domains. To achieve this goal, PEO-PPO amphiphiles were primarily modified with alkoxysilane moieties through the reaction of the terminal hydroxyl groups of the copolymer and 3-(triethoxysilyl)propyl isocyanate (IPTS)25. Then, ethoxysilane-modified PMs were prepared in water where hydrolysis resulted in a silanoldecorated surface that was cured by spray-drying. Due to the singular spraying mechanism of the Nano Spray-Dryer B-90 used in this work26, that is based on a vibrating mesh spray with holes in the 4-7 µm size range that produce ultra-fine droplets27,28, a novel kind of hybrid amphiphilic nanoparticle-in-nanoparticle nanomaterial was obtained (Scheme 1). These multimicellar carriers hosted high payloads of the hydrophobic model drug antiretroviral tipranavir (TPV) in the hydrophobic domains, and sustained the release with a more controlled zero-order fashion than the pristine counterpart that followed the classical biphasic release with an initial burst effect and a more moderate rate later on.

2. Results and discussion 2.1. From standard (physically instable) PMs to corona-crosslinked amphiphilic nanomaterials The aggregation pattern of thermo-sensitive PEO-PPO copolymers depends on the temperature, concentration, ionic strength, nature of the electrolyte and pH15. This behavior represents a hurdle to predict the nanostructure and to ensure the stability of the nanomaterials in the biological environment after administration. In addition, preclinical data demonstrated the limited ability of regular PMs to sustain the release. Thus, the exploration of friendly and green chemical pathways that confer physical stability without compromising the molecular structure of the cargo and that enable more controlled release kinetics are called for. The sol-gel chemistry represents a very versatile and clean approach to achieve this. In this work, we hypothesized that the conjugation of multifunctional alkoxysilane groups to the micellar


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corona of the PM and their later serial hydrolysis and condensation coupled to the spray-drying processing method will result in a novel kind of amphiphilic nanoparticle-in-nanoparticle (multimicellar) biomaterial with crosslinked siloxane domains that if designed and synthesized properly could be exploited to fine-tune the porosity and the diffusion properties of the nanomaterial and the release kinetics of the cargo (Scheme 1A).

2.2. Synthesis and characterization of ethoxysilane-capped copolymers The synthesis of the different derivatives was carried out by the reaction of the terminal -OH groups of PEO-PPOs with the isocyanate moiety of IPTS to render a urethane bond (Scheme 1B). To gain insight into the effect of the modification on micellization, copolymers with different molecular weight, architecture and hydrophilic-lipophilic balance (HLB) were used (Table 1). In general, the reaction yield was between 70% and 85%. IPTS moieties are very sensitive to moisture and thus, the modified copolymers were conserved under anhydrous conditions and low temperature (-20oC) to avoid hydrolysis that might result in partial crosslinking of the precursor before use. It is worth stressing that investigating the stability of the IPTS-modified copolymers was beyond the scope of this first work, though we did not observe any evidence of partial crosslinking in the powders during storage over several months. The chemical modification of the different copolymers was initially demonstrated by FTIR spectroscopy that showed a small characteristic peak at 1716 cm-1 (Figure S1) that belongs to the C=O stretching of newly formed urethane bonds. In addition, the absence of a band of the N=C=O stretching in the isocyanate moiety between 2200-2300 cm-1 indicated that unreacted IPTS residues were successfully removed from the product. 1H NMR spectra showed the presence of the characteristic signal of IPTS at 1.15 ppm (Figure S2), and the relative integration with the peaks of PPO was used to calculate the modification extent that ranged between 74% and 81% (Table 1). These results were supported by elemental analysis that evidenced the increase of the %N for all the IPTS derivatives with respect to the pristine counterpart and in good agreement with 1H NMR analysis (Table 1). Molecular weights and polydispersity indexes of pristine and modified copolymers were measured by gel permeation chromatography (GPC) (Table 1). Despite the relatively low molecular weight of IPTS (247.4 g mole-1), a clear increase of Mn and Mw consistent with the



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successful modification was observed; the increase was more pronounced for branched derivatives that could incorporate up to four IPTS residues per copolymer molecule than the linear ones that display two terminal reactive groups. In addition, the molecular weight increase and the negligible change in the polydispersity confirmed that the copolymers did not undergo partial polymerization through the condensation of silanol groups generated during the reaction due to moisture traces; conduction of the modification reaction under non-dry conditions resulted in massive crosslinking of the mixture. The IPTS modification was further confirmed by thermal analysis (DSC) where the modification reduced the melting temperature (Tm) and enthalpy (∆Hm) of PEO blocks in all the derivatives between 4 °C and 9 °C and by 14-22%, respectively (Table S1). The effect was stronger on T904, a copolymer of lower molecular weight and PEO content and greater relative w/w IPTS modification than the other two derivatives. Then, the weight loss (%) under heating was measured by TGA. All the pristine and IPTS-modified counterparts presented one main degradation stage (Figure S3). As expected, IPTS-capped copolymers exhibited the degradation at a lower T than the unmodified counterparts due to the greater thermal instability of ethoxysilane bonds that usually undergo decomposition at 0.990). Copolymer solutions were used as blank. Then, the solubility factor (fs) determined according to Equation 4 + =

, (4) -

Where Sa is the apparent solubility of TPV in the PMs and Si is its intrinsic solubility in a polymer-free medium (8 µg mL-1), at 37 °C. The thermal properties of TPV-loaded micelles (unmodified and modified) after spray-drying were analyzed by DSC and the size and size distribution by DLS, as depicted above. 4.2.9. Drug release The release of TPV from pristine and corona-crosslinked PMs was assessed employing phosphate buffered saline (0.05M PBS and pH 7.3) release medium prepared with potassium phosphate dibasic (K2HPO4, Spectrum chemical MFG Corp., Gardena, CA, USA) and monobasic (KH2PO4, EMD Millipore corp., Billerica, MA, USA). Ten percent TPV-loaded PMs were diluted (1/50) in water pre-heated to 37 °C to a final copolymer concentration of 0.2% w/v and of drug in the 300340 µg mL-1 range. Then, 5 mL of this dilution was placed in a dialysis membrane (regenerated cellulose tubular membrane, molecular weight cut off = 3500 Da) and immersed in 1.5 L of release medium at 37 °C. These volumes ratio ensured sink conditions. Aliquots of release medium (30 mL) were removed at predetermined time intervals and replaced by fresh pre-heated (37 °C) PBS to keep the total volume constant. The drug concentration was monitored by UV-Vis spectrophotometry after extraction from the aqueous medium with DCM (3 x 30 mL, 1 min) evaporated at room temperature and normal pressure, and re-dissolution in methanol (1 mL). The release experiment from crosslinked PMs was performed using the same method, though by directly preparing a 0.2% w/v micellar suspension in water. The final TPV concentration was similar to the one in the unmodified PMs. Assays were carried out in triplicates and results expressed as mean ± S.D. Average release data was fitted to different models using the DDSolver Software 1.0, a free calculation program used to analyze dissolution or fit drug release data44. For Korsmeyer-Peppas model, only data between 0% and 60% release was considered. 4.2.10. Cell compatibility The cell compatibility of crosslinked F127 PMs was evaluated in a model of intestinal epithelium, Caco2 cell line (ATCC® HTB-37TM, ATCC®, Manassas, VA, USA). Cells were


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cultured in Eagle's Minimum Essential Medium (EMEM, Life Technologies Corp.) supplemented with L-glutamine, 10% heat-inactivated fetal bovine serum (FBS, Sigma-Aldrich) and penicillin/streptomycin (5 mL of a commercial mixture of 100 U/mL de penicillin + 100 µg /mL streptomycin per 500 mL medium, Sigma-Aldrich) and maintained at 37ºC in a humidified 5% CO2 atmosphere and split every 4-5 days. Cells were harvested by trypsinization (trypsin-EDTA 0.25%, Sigma-Aldrich) and the number of live cells quantified by the trypan blue (0.4%, SigmaAldrich) exclusion assay. To determine the compatibility, cells were cultured in 96-well plates (7.5 x 103 cells/well) and allowed to attach for 5 days. Then, the culture medium was replaced by fresh medium, and the corresponding volume of pristine F127 or crosslinked F127-IPTS PMs sample of concentration 1% w/v PBS (pH = 7.4, sterilized by filtration through 0.22 µm filter) was added to result in a final concentration of 0.1% w/w (final medium volume of 200 µL). At 4 h, the medium was removed and fresh medium (100 µL) and 25 µL of sterile MTT solution (5 mg/mL, Sigma-Aldrich) were added. Samples were incubated for 4 h (37oC, 5% CO2), the supernatant was removed, the formazan crystals dissolved with DMSO (100 µL), solutions transferred to a new 96-well plate and the absorbance measured at 530 nm (with reference of 670 nm) in a Multiskan GO Microplate Spectrophotometer with SkanltTM software (Thermo Fisher Scientific Oy, Vantaa, Finland). The percentage of live cells was estimated with respect to a control treated only with culture medium that was considered 100% viability. Results are expressed as mean ± S.D. Statistical analysis was performed by t-test on raw data (Excel, Microsoft Office 2013, Microsoft® Corporation).

Supporting Information The Supporting Information is available free of charge on the ACS Publications website. FTIR and 1H-NMR spectra of F127 copolymer before and after modification with IPTS; thermal analysis of pristine and IPTS-modified PEO-PPO copolymers (DSC and TGA); size and size distribution of 2% w/v polymeric micelles made of pristine and IPTS-capped PEO-PPOs (DLS); SEM and TEM analysis of pristine and modified polymeric micelles; fitting of average release data from TPV-loaded pristine and IPTS-crosslinked F127 polymeric micelles (DDSolver Software 1.0); Caco2 cell viability upon 4 h exposure to 0.1% w/v pristine F127, non-crosslinked



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F127-IPTS and crosslinked F127-IPTS PMs (MTT assay); feeding ratios of PEO-PPO copolymer, catalyst and IPTS to synthesize ethoxysilane-capped derivatives. Acknowledgements. Authors thank the financial support of Technion (internal grant #2019300) and the European Union's - Seventh Framework Programme under grant agreement #612765MC-NANOTAR. IS thanks partial scholarship support by the RBNI.

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Figures A

-

O-CH2-CH3 group

B

(

)(

)(

)

99

67

99

2

+

F127

IPTS 75°C 1h

SnOct2

(

)(

)(

)

99

67

99

H2O (Hydrolysis)

)(

(

99

EtOH

)(

)

67

99

Spray-drying or freeze-drying (Condensation)

(

H2O

)(

)(

)

99

67

99

34

ACS Paragon Plus Environment

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Scheme 1. (A) The design rationale behind the Nanoparticle-in-Nanoparticle hybrid material based on the corona-crosslinking of PEO-PPO micelles by sol-gel chemistry coupled to spraydrying and the generation of crosslinked inorganic (poly(siloxane)) rate-controlling domains. (B) Synthetic pathway for the synthesis of F127-IPTS and the production of crosslinked F127-IPTS multimicellar nanomaterials by means of the sol-gel process.



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A

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B

2 µm

20 µm

2 µm

20 µm

Figure 1. HR-SEM micrographs of spray-dried (A) pristine F127 and (B) F127-IPTS PMs. Analysis was conducted on the dry powders.

Figure 2. Characterization of 2% w/v spray-dried F127-IPTS PMs. (A) Histogram of size distribution (%Intensity) of F127-IPTS PMs after spray-drying and re-dispersion to a final 1% w/v concentration at 23 °C, as measured by DLS, (B) HR-SEM micrograph of 1% w/v F127IPTS after spray-drying, re-dispersion and casting on carbon tape and (C) cryo-TEM micrographs of 1% w/v F127-IPTS PMs of 1% w/v F127-IPTS PMs before (left) and after (right) spray-drying


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of a 2% w/v micellar system. Original micelles (left) were rapidly frozen from 37 °C, while spray-dried counterparts (right) were re-dispersed in water at 23oC before the analysis.

A

D

B

E

C

F

Longer irradiation time

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Figure 3. STEM micrographs of drug-free F127-IPTS PMs before (A-C) and after (D-F) spraydrying at different irradiation time points.

A

B

2 µm

20 µm

20 µm

Figure 4. HR-SEM micrographs of spray-dried 2% w/v TPV-loaded (A) pristine F127 and (B) F127-IPTS PMs. Arrows in A point out drug crystals.

A

B

Intensity (%)

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Size (d. nm)

Figure 5. Characterization of 2% w/v spray-dried TPV-loaded F127-IPTS PMs. (A) Histogram of size distribution (%Intensity) F127-IPTS PMs after spray-drying and redispersion to a final of


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1% w/v concentration at 23 °C, as measured by DLS and (B) HR-SEM micrograph of 1% w/v F127-IPTS after spray-drying cast on carbon tape.

Figure 6. Cryo-TEM micropgraphs of TPV-loaded F127-IPTS PMs. (A) 5% w/v micellar dispersion (drug content of 6.25 mg mL-1) before spray drying and (B) 1% w/v dispersion (drug content of 1.25 mg mL-1) after spray drying. The micellar concentration in all the spray-dried samples was 2% w/v.



90 80

Cumulative TPV release (%)

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70 60 50 40 30 20 10 0 0

5

10

15

20

25

Time (h) Figure 7. Cumulative TPV release from pristine F127 (●) and crosslinked F127-IPTS (■) PMs, over 24 h.


Amphiphilic Nanoparticle-in-Nanoparticle Drug ... - ACS Publications

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