Inverse Flash NanoPrecipitation for Biologics Encapsulation

Chapter 12

Downloaded by UNIV OF FLORIDA on January 7, 2018 | http://pubs.acs.org Publication Date (Web): November 15, 2017 | doi: 10.1021/bk-2017-1271.ch012

Inverse Flash NanoPrecipitation for Biologics Encapsulation: Understanding Process Losses via an Extraction Protocol Chester E. Markwalter and Robert K. Prud’homme* Department of Chemical and Biological Engineering, Princeton University, Princeton, New Jersey 08544, United States *E-mail: [email protected].

Inverse Flash NanoPrecipitation (iFNP) is a scalable technique for encapsulating hydrophilic molecules such as peptides and proteins in nanoparticles at high loadings. These nanoparticles, which have a hydrophilic core and hydrophobic corona, may be incorporated directly into microparticles for sustained release applications. Use of iFNP instead of the traditional double emulsion process can enable higher loadings and encapsulation efficiencies. We have developed an extraction-based protocol to rapidly evaluate the impact of iFNP formulation parameters on process losses without the added complexity of producing microparticles. Among other parameters, the external osmotic pressure and, for larger biologics, the crosslinking density were found to impact the extraction process. Microparticles were subsequently produced with a target loading of 25% of a model biologic at greater than 90% encapsulation efficiency using these insights.

Introduction The Need for Improved Biologics Delivery Vehicles Biologically-derived drugs, or biologics, comprise a class of therapeutics which is rapidly growing in importance. These water-soluble drugs include peptides and proteins, which have a more complex structure than small molecule drugs. In 2016, they accounted for 31% of FDA approvals for novel molecules © 2017 American Chemical Society Ilies; Control of Amphiphile Self-Assembling at the Molecular Level: Supra-Molecular Assemblies with Tuned ... ACS Symposium Series; American Chemical Society: Washington, DC, 2017.


(1). By 2019, the global market is expected to surpass $200 billion. Such growth is a result of the high potency and efficacy of biologics coupled with their improved tolerability and safety profiles (2). A major barrier to the more widespread use of biologics is the challenge of successfully administering them to patients. One of the hurdles posed by biologics is their rapid clearance from the bloodstream, with serum half-lives sometimes measured on the order of minutes (3). The physical properties of biologics also restrict them from oral administration because they are poorly permeable across membranes and can be degraded in the gastrointestinal tract (3). Consequently, biologics are often administered by frequent injections. Reducing injection frequency from daily to weekly or even monthly with sustained release formulations can have both medical and economic benefits. Adverse events can be reduced and improved compliance can raise patients’ health outlook with a concomitant reduction in avoidable costs (4). In the US, the cost of noncompliance (not limited to biologics use) is on the order of $100 billion per year (5). Sustained release formulations of biologics have been in existence since the 1970s. The most widely used formulations consist of microparticles formed by a hydrophobic polymer scaffold, such as poly(lactic-co-glycolic acid) (PLGA), with the biologic incorporated into pores within this structure (6). There are examples of marketed formulations, such as the Lupron Depot, which received FDA approval in 1997. However, success with such formulations has been limited by manufacturing shortcomings. The most common production method uses a double emulsion process where a primary aqueous phase containing the biologic is emulsified in an organic solvent containing the hydrophobic scaffold polymer. This primary emulsion is then emulsified in a second aqueous phase. Each emulsion is stabilized by surfactant or polymer additives. The organic solvent may then be stripped out, collapsing the hydrophobic polymer around the primary aqueous phase and producing biologic-containing pores (7). The double emulsion process suffers from unavoidable trade-offs between high loading (biologic content as a fraction of total solids in the formulation) and encapsulation efficiency (the fraction of biologic lost to the external aqueous phase during processing). These two metrics are important in defining the economic feasibility of a formulation. High loadings decrease the amount of excipients required, while a high encapsulation efficiency (EE) indicates low losses of an expensive drug during processing. In the double emulsion process, high biologic loading increases the likelihood of pore contact with the oil-external water interface, resulting in biologic loss from the microparticle and lower encapsulation efficiency. Alternatively, increasing scaffold polymer levels to reduce the likelihood of this occurring produces formulations which have ineffectually low loadings and undesirable release characteristics, such as dormant periods and incomplete release (7). The optimal microparticle formulation for sustained release would contain high loadings of biologic in small pores that are uniformly dispersed throughout the microparticle. This would allow for small injection quantities and a clinically-relevant release profile. A process which can do this with high encapsulation efficiency would be an economical approach to sustained biologics delivery (7). 276 Ilies; Control of Amphiphile Self-Assembling at the Molecular Level: Supra-Molecular Assemblies with Tuned ... ACS Symposium Series; American Chemical Society: Washington, DC, 2017.


Inverse Flash NanoPrecipitation for Biologics Encapsulation Recently, our lab described a novel technique for encapsulating hydrophilic compounds such as biologics inside nanoparticles (NPs), which can then be incorporated into microparticles (7). The process, inverse Flash NanoPrecipitation (iFNP), is shown schematically in Figure 1. It is a modification of Flash NanoPrecipitation, which has been extensively described (8–10). Both techniques use confined impinging jet mixers to achieve rapid micromixing of two streams: a solvent stream containing a compound of interest with an amphiphilic block copolymer (BCP) and an antisolvent stream. The rapid change in solvent quality results in uniform nucleation of the compound and collapse of one polymer block. Nanoparticle formation begins as nuclei form and aggregate. Particle growth is halted by assembly of the collapsed polymer block onto the particle surface, with the polymer brush of the non-collapsed block forming a stabilizing corona around the nanoparticle. In iFNP, a hydrophilic compound is precipitated into the NP core by a lipophilic antisolvent. A typical non-degradable BCP would be poly(acrylic acid)-b-poly(styrene) (PAA-b-PS) while a suitable degradable polymer is poly(aspartic acid)-b-poly(lactic acid). The NP corona consists of the hydrophobic block of the BCP while the core contains the hydrophilic polyacid. Since this architecture is the opposite of traditional NPs, we will refer to them here as “inverted” or “inverse” NPs. In order to stabilize the core of the inverted NPs against swelling or bursting upon exposure to aqueous interfaces, we employ ionic crosslinking of the acid residues of the hydrophilic block. Multivalent cations such as Fe3+ or polyamines highly efficiently stabilize the inverted NPs against favorable solvents for the BCP (11). This crosslinker is added into the antisolvent stream during the iFNP process such that “1 equivalent” represents a total crosslinker ionic charge equaling the number of ionizable groups on the hydrophilic block. A hydrogel forms from the electrostatic interactions between the polymer acid groups and the positively charged species. After iFNP, the biologic is dispersed in an organic solvent in NPs on the order of 100nm in size. This composition mirrors that of the primary emulsion in the microparticle synthesis described above, except that our inverted NPs may be thought of as forming a nanoparticle-in-oil rather than water-in-oil primary emulsion. In the second processing step, microparticles are formed by adding the hydrophobic scaffold polymer to the organic phase, producing an emulsion in an external aqueous phase, and then stripping the organic solvent from the sample. There are three advantages of iFNP over the traditional double emulsion process (7). First, the BCP stabilizes the fine dispersion within the microparticle against coalescence. Second, the BCP provides a barrier to biologic loss during processing, which enables high microparticle EE even at high loadings. Finally, the inverted NP architecture provides an additional means to tune release of the biologic from the microparticle through the NP shell degradation. With iFNP, we are able to achieve loadings in the inverted NP of 50%-90% (7). Microparticles where the scaffold polymer content is 25 wt% have been produced from the NP-in-oil stream, affording overall loadings that are an order of magnitude higher than typical double emulsion values. The two step process in 277 Ilies; Control of Amphiphile Self-Assembling at the Molecular Level: Supra-Molecular Assemblies with Tuned ... ACS Symposium Series; American Chemical Society: Washington, DC, 2017.


Figure 1 can produce the desired microparticle morphology of a finely dispersed biologic while maintaining the economic feasibility and process scalability necessary for translation to the clinic.

Figure 1. (A) There are two steps to highly loaded microparticles: inverse Flash NanoPrecipitation and the incorporation of the inverted NPs into polymeric microparticles. Note the length scales for each particle type. (B) The schematic of the extraction protocol employed to compare inverted NP formulations prior to microparticle production. Process Understanding from a Novel Extraction Protocol We seek to limit processing losses during the microparticle formation step to achieve high EE. Some of the factors impacting encapsulation efficiency were already discussed above, while a more detailed treatment from our group may be found in the literature (7). We believe the inverted NP architecture will improve EE because it provides additional barriers against diffusion into the external aqueous phase, which may occur prior to removal of the organic solvent. (After solvent stripping, the hydrophobic polymers are in a collapsed state and biologic release becomes a degradation-controlled process.) We sought an assay to assess the diffusion barriers presented by the inverted NPs without the added complexities of forming microparticles. (This complexity includes variability in the stream compositions, the emulsion process, and the solvent removal process.) To this end, an extraction protocol, where the nanoparticles-in-oil phase is continually contacted with an aqueous phase, can 278 Ilies; Control of Amphiphile Self-Assembling at the Molecular Level: Supra-Molecular Assemblies with Tuned ... ACS Symposium Series; American Chemical Society: Washington, DC, 2017.


serve as an approximation of the conditions that lead to process losses during microparticle formation. This protocol is shown schematically in Figure 1B and is later described in additional detail. The assay differs from the microparticle process itself in two ways. First, it lacks stabilizing additives necessary to form a stable emulsion. Second, while microparticle EE is determined at a finite time during processing, this extraction assay follows release kinetics over time into the aqueous phase. We expect the release rates measured in this extraction protocol to parallel the EE trends in microparticle processing while not accurately representing absolute values. With this assay, faster release into the aqueous phase indicates reduced protection of encapsulated material. We also sought an affordable model biologic system that would allow us to vary molecular weight and net charge without changing other physical properties. We wanted a system that would balance low materials costs and highly sensitive detection limits. In this report, we describe the model biologics, fluorescently-tagged polysaccharides, and demonstrate the diffusional barrier provided by the BCP against process losses. Based on the extraction findings, we produce microparticles with high loadings and encapsulation efficiencies using inverted NPs produced by iFNP. There is on-going work to extend this process to proteins, including the evaluation of processing effects on secondary structure.

Materials and Methods Materials Maltodextrin DE 4-7 (3k MD), Maltodextrin DE 16.5-19.5 (1k MD), technical grade tetraethylenepentamine (TEPA), 7-amino-4-methyl coumarin (AMC), sodium cyanoborohydride (NaCNBH3), chloroacetyl chloride, potassium iodide, anhydrous ethyl acetate, polystyrene (Mn: 170,000, Mw: 350,000), poly(vinyl alcohol) 98% hydrolyzed (Mw: 11,000-31,000), and sodium chloride (NaCl) were purchased from Sigma-Aldrich (St. Louis, MO). Dextran T20 (Dex) was purchased from Pharmacia Fine Chemicals (Uppsala, Sweden). Optima ® chloroform (CHCl3), ACS grade glacial acetic acid, and HPLC grade dimethylsulfoxide (DMSO) were purchased from Fisher Scientific. Poly(acrylic acid)-b-poly(styrene) (4.8k-b-5k PDI 1.4, 4.3k-b-15k PDI 1.15, and 5.5k-b-52k PDI 1.08) was purchased from Polymer Source (Dorval, Quebec). Anhydrous powdered sodium hydroxide was purchased from MP Biomedicals (Santa Ana, CA). All reagents were used as received. Deionized water was treated to a resistivity of 17.8mΩ-cm or greater (NANOpure Diamond, Barnstead International, Dubuque, IA). Fluorescent Modification of Polysaccharides Maltodextrin (MD) and Dextran (Dex) of different molecular weight ranges were modified with 7-amino-4-methylcoumarin (AMC) via either a reductive amination route (Scheme 1) or a chloroacetyl chloride-mediated route (Scheme 2). In Scheme 1, MD reagents with average molecular weights of approximately 1000 g/mol and 3275 g/mol will be referred to as 1k MD-AMC 279 Ilies; Control of Amphiphile Self-Assembling at the Molecular Level: Supra-Molecular Assemblies with Tuned ... ACS Symposium Series; American Chemical Society: Washington, DC, 2017.


and 3k MD-AMC, respectively. Dex had an average molecular weight of 20,000 g/mol and the fluorescent product will be referred to as 20k Dex-AMC. In Scheme 2, the corresponding MD products will be referred to as 1k MD-amide-AMC and 3k MD-amide-AMC. Dex-amide-AMC was not produced. In Scheme 1, polysaccharide, AMC, and sodium cyanoborohydride were dissolved in DMSO containing 30 vol% glacial acetic acid according to the conditions summarized in Table 1 (12). The coupling reaction was carried out protected from light with stirring at 60 °C for 2 hours. After cooling, the polysaccharide was precipitated with 14 volumes of methanol. After redissolving the solids in water to a final concentration of 40-50 mg/ml, the polysaccharide was reprecipitated in methanol (final solvent composition greater than 85% methanol) and the solids were rinsed with acetone to remove residual AMC. The solids were dried under vacuum at room temperature and then characterized. In Scheme 2, AMC (155 mg) was first reacted with chloroacetyl chloride (2.07 eq.) in 4 ml ethyl acetate at 70 °C for 2 hours under nitrogen (13). The white slurry was isolated and rinsed with ethyl acetate before drying under vacuum to afford AMC-Cl.

Scheme 1. Synthesis of MD-AMC by reductive amination. Dex-AMC is produced by an analogous route.

280 Ilies; Control of Amphiphile Self-Assembling at the Molecular Level: Supra-Molecular Assemblies with Tuned ... ACS Symposium Series; American Chemical Society: Washington, DC, 2017.


This product was then randomly coupled via an ether linkage to 1k and 3k MD. Briefly, the polysaccharide was dissolved in DMSO followed by addition of a large excess of powdered sodium hydroxide with rapid stirring. AMC-Cl (1k MD – 1 eq.; 3k MD – 2.2 eq.) and potassium iodide (1k MD – 0.7 eq.; 3k MD – 1.6 eq) were then added. This mixture was reacted for 45 minutes at ambient conditions. The sodium hydroxide was pelleted by centrifugation and the solution was recovered by decanting. The solution was then acidified with acetic acid. The product was precipitated with 17.5 volumes of methanol and rinsed with additional methanol. The solids were dissolved in deionized water at around 100 mg/ml and precipitated with 30 volumes of methanol. The solids were rinsed with acetone and then dried under vacuum at room temperature.

Scheme 2. Two-step synthesis of MD-amide-AMC. AMC-Cl is stochastically conjugated along the chain length in this route, represented by unspecified chain lengths on either side of the modified glucose monomer.


Table 1. Summary of the reaction conditions used in the synthesis of MD-AMC and Dex-AMC via Scheme 1 (reductive amination) Polysaccharide (mg)

Concentration (mg/ml)

AMC (eq.)

NaCNBH3 (eq.)

1k MD

670

36

3

25

3k MD

333

20

10

25

20k Dex

250

36

10

25


Characterization of AMC-Modified Polysaccharides The labeled polysaccharides were characterized using thin layer chromatography (TLC) to confirm successful conjugation and to detect residual AMC or AMC-Cl. Additionally, nuclear magnetic resonance (NMR) was used to confirm successful conjugation. Proton NMR analysis was carried out using a Bruker AVANCE III (500 MHz) spectrometer with a CryoProbe optimized for 1H detection. An internal standard of 4,4-dimethyl-4-silapentane-1-sulfonic acid (DSS) at 0.01 mg/ml in D2O (Cambridge Isotopes Laboratory) was used to calculate the AMC concentration by integration against the AMC doublet at 7.54 ppm. The AMC weight fraction was found using the total solids concentration in the NMR sample. The reaction conversion was determined based on the theoretical AMC weight fraction for a completely modified polysaccharide sample, which would contain a single fluorophore per chain. Fluorescence measurements were carried out using a Molecular Devices SpectraMax i3x plate reader at the excitation maxima for each product. Inverted Nanoparticle Formation with iFNP Inverted NPs were produced by iFNP using a Confined Impinging Jet (CIJ) mixer (8). The hydrophilic stream (DMSO/THF/Water mixture) contained the fluorescent polysaccharide at 5 mg/ml and the PAA-b-PS block copolymer at varying concentrations. The block sizes for each formulation are summarized in Table 2. The hydrophobic stream (CHCl3) contained the crosslinker, TEPA, added at a defined charge equivalents with respect to the total ionizable groups in the PAA block. The formulation compositions are summarized in Table 2. Formulations with larger PS block molecular weights were adjusted to account for the relative decrease in PAA content of the BCP. The two streams (500 μL each) were rapidly mixed using the CIJ mixer and collected in a stirred scintillation vial containing 4.5 ml of CHCl3. Particle size was characterized by Dynamic Light Scattering (DLS) using a Zetasizer Nano ZS (Malvern, Worcestershire, UK) at 25°C by diluting ten-fold with CHCl3. Size distribution was determined from CONTIN analysis and polydispersity (PDI) from a cumulants analysis of the data. To confirm TEPA crosslinking of the PAA chains, DMSO was employed as a diluent. Without crosslinker, the inverted NPs dissolve in this diluent and are not detected by DLS. Control samples consisted of an identical formulation that did not contain the BCP. 282 Ilies; Control of Amphiphile Self-Assembling at the Molecular Level: Supra-Molecular Assemblies with Tuned ... ACS Symposium Series; American Chemical Society: Washington, DC, 2017.

Table 2. Formulation summary for inverted NPs produced by iFNP Polysaccharide

PAA-b-PS (g/mol*10-3)

DMSO/THF/ Water %

[BCP] (mg/ml)

Loading (wt%)

1k MD

4.8-b-5

50/45/5

5

50%

4.3-b-15

50/45/5

10

33%

5.5-b-52

50/47/2

15

25%

4.8-b-5

50/45/5

5

50%

4.3-b-15

50/45/5

10

33%

5.5-b-52

50/47/2

15

25%

4.8-b-5

90/0/10

5

50%

4.3-b-15

80/10/10

10

33%


3k MD

20k Dex

Release Kinetics Determined via Extraction To each solution of inverted NPs in CHCl3, 5 ml of 13 wt% sodium chloride in water was added slowly. This brine concentration was selected after evaluating the extraction at a range of compositions to balance reduced losses with amount of salt required. This biphasic mixture was placed on a rotary mixer at constant speed. For each time point, the vial was removed from the rotary mixer, the biphasic mixture was transferred to a conical tube and centrifuged at 1000g for 1-3 minutes. The brine layer was sampled and stored at 2-8 °C until analysis. An equal volume of fresh brine was added to the vial and then it was returned to the rotary mixer. This process is summarized in Figure 1B. Sample fluorescence was analyzed in duplicate using the SpectraMax i3x plate reader with the excitation wavelength set to the fluorophore maximum. Each formulation was prepared and analyzed in triplicate. The fraction released was determined as compared to the average brine concentration for a control sample prepared as described above. Note that the control sample concentration in the aqueous phase was constant across the full experiment, indicating rapid and complete extraction of unprotected polysaccharides in this protocol. Microparticle Formation Two formulations were selected for microparticle formation studies. Both consisted of PAA4.8k-b-PS5k with either 3k MD-AMC or 20k Dex-AMC encapsulated. The inverted NP solution in chloroform was extracted for 30 minutes to 2 hours with an equal volume of 13 wt% brine solution to remove DMSO and unencapsulated biologic. Polystyrene (Mn 170,000) in chloroform was added to the inverted NP solution after separation of the aqueous phase such that the polystyrene mass was equivalent to the total inverted NP mass. The chloroform solution was then concentrated by rotovap to a total mass concentration of 10 mg/ml. Particle size stability was confirmed by DLS following processing. To produce microparticles, 75 μL of organic phase was added to 10 ml 283 Ilies; Control of Amphiphile Self-Assembling at the Molecular Level: Supra-Molecular Assemblies with Tuned ... ACS Symposium Series; American Chemical Society: Washington, DC, 2017.

of an aqueous phase comprised of 1 wt% poly(vinyl alcohol) with or without 7.5 wt% NaCl. Hand mixing afforded small droplets dispersed in the aqueous phase. The chloroform was then removed by rotovap. The supernatant was sampled after centrifugation at 2000g and the fluorescence intensity of unencapsulated material determined with a Hitachi F-7000 Fluorescence Spectrophotometer. The encapsulation efficiency was determined in reference to the polysaccharide content assayed by complete aqueous extraction of a control sample which contained no BCP.


Results and Discussion Fluorescently-Labeled Polysaccharides as a Model Biologic System Polysaccharides of various molecular weights were selected as model biologics for release studies from inverted NPs because they afford a number of benefits. They lack secondary structure, allowing for isolation of charge and molecular weight effects. This permits a direct comparison of release kinetics without other variables confounding the analysis. Later studies will probe the behavior of proteins in this system. Polysaccharides are cheap, facilitating a large number of studies without the costs associated with peptides and proteins as models. The attachment of a bright, photostable fluorophore such as AMC enables detection of very low concentrations of the polysaccharide (sub-μg/ml levels) that would be unobtainable with traditional polysaccharide analytical techniques (14). In Scheme 1, amine linkages to a single fluorophore were generated via the reducing end of the polysaccharide. This afforded bright fluorescent sugars with a characteristic excitation maximum at 365nm and an emission maximum at 450 nm. No residual AMC was observed by TLC. NMR analysis in D2O afforded the expected spectra for the aryl protons of AMC while analysis in DMSO-d6 did not detect the exchangeable protons for the unconjugated amine in AMC. The yields and conversions for Scheme 1 are summarized in Table 3. For this route, conversion represents the fraction of polysaccharide chains modified via the reducing end with a single fluorophore. Increased yields and conversions for larger polysaccharides are due, respectively, to more efficient precipitation conditions and to the higher AMC equivalents which were feasible. In Scheme 2, the initial modification of AMC with chloroacetyl chloride afforded an amide linkage with the alkyl halide and was completed in 83% mass yield. 1H and 13C NMR analysis confirmed the expected product was obtained quantitatively. Coupling to the polysaccharide via alkoxide attack on the alkyl halide of AMC-Cl afforded acceptable yields and conversions, as summarized in Table 3. The excitation maximum was shifted to 325 nm and the emission maximum to 395nm, an additional confirmation of the amide linkage. No unconjugated AMC-Cl was observed by TLC. 1H NMR analysis in D2O afforded the expected spectra and integration against DSS provided the conversion value. For Scheme 2, this value represents the fraction of polysaccharide chains modified by the fluorophore, assuming a single modification per chain. In this synthetic approach, each polysaccharide was randomly modified via ether 284 Ilies; Control of Amphiphile Self-Assembling at the Molecular Level: Supra-Molecular Assemblies with Tuned ... ACS Symposium Series; American Chemical Society: Washington, DC, 2017.

linkage. Low conversions were maintained to avoid significantly modifying the hydrophilic nature of the polysaccharide and thus justify the assumption of a single fluorophore modification per chain.

Table 3. Reaction performance summary


Scheme 1

Scheme 2

Polysaccharide

1k

3k

20k

1k

3k

Mass Yield

27%

69%

87%

21%

50%

Conversion

9%

21%

74%

11%

5%

High Reproducibility of Inverted NPs Generated by iFNP To form inverted NPs with the AMC-tagged polysaccharides, an amine-based crosslinker was selected rather than a multivalent cation. This avoided the potential formation of metal complexes reported in the literature for certain coumarin derivatives (15, 16). Complex formation conceivably would result in modified release kinetics of the model biologic and affect the fluorescence signal. Tetraethylenepentamine (TEPA) afforded strong, tunable crosslinking without the concerns regarding coumarin interactions. Inverted NPs made using iFNP were highly reproducible. Table 4 presents the NP size and standard deviation for three preparations of each formulation. The 20k Dex-AMC NPs show a notable increase in size over the smaller polysaccharides. This primarily reflects the impact of higher water content used in the formulation. The average polydispersity of each formulation is also summarized. No formulation had a polydispersity above 0.20, indicating a relatively monodisperse population for all samples (17). This characteristic is desirable for microparticle applications because it enables uniform “pore” size, reducing inhomogeneity across the microparticle volume. All formulations were found to be stable against swelling in DMSO, indicating strong crosslinking by TEPA. Inverted NPs formed from MD-amide-AMC and PAA4.8k-b-PS5k were similar to the MD-AMC particles. For the 1k MD-amide-AMC, particle size was 69 ± 2 nm with a PDI of 0.06. For the 3k MD-amide-AMC, particle size was 70 ± 3 nm (PDI of 0.08). We therefore do not expect that differences in NP architecture would contribute to release kinetics differences. The EE reported in Table 4 was calculated from the brine phase content sampled after 30 minutes of contact time. For this time point, control samples had reached the equilibrium value in the brine phase (data not shown). Thus, this point represents full extraction of unencapsulated material with minimal error due to slower release of encapsulated biologic. All encapsulation efficiencies were greater than 97%, demonstrating low process losses. Further confirmation of encapsulation within inverted NPs is seen in the distinct size differences between formulations which contain the BCP and the controls (micron-scale aggregates for 1k and 3k MD-AMC; 230nm unstable particles for 20k Dex-AMC). 285 Ilies; Control of Amphiphile Self-Assembling at the Molecular Level: Supra-Molecular Assemblies with Tuned ... ACS Symposium Series; American Chemical Society: Washington, DC, 2017.

Table 4. Inverted NP formulation characterization by DLS and extraction

1k MD-AMC


3k MD-AMC

20k Dex-AMC

PS Block Size (g/mol)

NP size (nm)

PDI

EE

5,000

62 ± 3

0.10

97.8%

15,000

81 ± 5

0.20

98.9%

52,000

66 ± 6

0.17

99.3%

5,000

64 ± 2

0.07

97.4%

15,000

89 ± 4

0.20

98.9%

52,000

61± 1

0.11

99.4%

5,000

147 ± 3

0.14

98.7%

15,000

156 ± 4

0.14

98.8%

iFNP affords essentially quantitative encapsulation of the biologic at loadings of 50% across a range of biologic molecular weights. While additional processing steps are required to produce a microparticle, high loading and EE in the final formulation demands that each processing step be highly efficient as well. The benefit of BCP encapsulation in reducing process losses is readily apparent from the EE data. As noted above, unencapsulated control samples are rapidly extracted while encapsulated material is protected from high losses. This observation provided further incentive to study the diffusive barrier presented by inverted NP formulation parameters in a systematic fashion.

Salt Content of Aqueous Phase Impacts Release The impact of brine content in the aqueous phase on the extraction of 1k MD-AMC was evaluated after 10 minutes of contact time. The use of 15wt% brine leads to a 5-fold reduction in the fraction of MD-AMC extracted at short times over deionized water, as seen in Figure 2. Above this brine content, the effect becomes diminished. The reduction in extracted amount (or more properly, the rate of extraction) is an osmotic pressure effect from reduced PAA hydrogel swelling (18). If swelling occurred, the dense polystyrene corona would be disrupted by the core expansion. This would result in unstable particles as the core becomes exposed to external solvent and would permit more rapid losses of encapsulated material. This effect was observed experimentally. With deionized water, a film develops at the interface, whereas no such film develops with a brine phase. This film is indicative of unstable NPs at the interface. Future work will evaluate whether similar effects are observed with other osmolytes, such as small carbohydrates.



Figure 2. Impact of aqueous phase brine content on the extraction of 1k MD-AMC after 10 minutes of contact time. Crosslinking Strength Impacts Release Rates for Large Biologics By varying the equivalents of crosslinker in the antisolvent stream, we are able to tune the extent of PAA hydrogel crosslinking. This effect can be evaluated by DLS using DMSO as a diluent. With extensive crosslinking, we observe a strong correlation signal and minimal size change. With sparse crosslinking, we observe particle swelling and a weaker correlation signal intensity because the solvent-swollen core has a low refractive index contrast with the external solvent phase. For 1k MD-AMC, the inverted NP size increase in changing from a CHCl3 to DMSO diluent is 5%, 42%, and 98% for 1 eq., 0.5 eq., and 0.15 eq. of TEPA, respectively. The DLS correlation signals, which show decreased intensity with lower crosslinker equivalents, are plotted in Figure 3. Since release depends on diffusion of the biologic through this mesh, theory would predict that slower kinetics would be observed as the biologic hydrodynamic radius approaches that of the hydrogel mesh (19). A tighter hydrogel mesh in the core of the inverted NPs, produced through greater crosslinker equivalents, would decrease release rates. We do observe this decreased release rate from our inverted NPs, as shown in Figure 4. 1k MD-AMC showed no difference across the full range of crosslinker equivalents, while the 20k Dex-AMC showed a 30% decrease in rate when TEPA equivalents were increased from 0.5 to 1. No changes in NP size or strength of crosslinking (as determined by DLS analysis, data not shown) were seen over the course of the experiment. If the changes to crosslinking of the hydrogel resulted in differences in NP interactions at the liquid-liquid interface, we would have observed changes in release kinetics for the 1k MD-AMC. Instead, PAA hydrogel crosslink density effects were detected only upon an increase in hydrodynamic radius from around 1.1 nm to 3.3 nm for the 1k MD and the 20k Dex, respectively (20). We therefore expect extent of crosslinking to serve as a barrier to diffusion only for larger peptides or proteins. 287 Ilies; Control of Amphiphile Self-Assembling at the Molecular Level: Supra-Molecular Assemblies with Tuned ... ACS Symposium Series; American Chemical Society: Washington, DC, 2017.


Figure 3. DLS correlation functions for 1k MD-AMC inverted NPs crosslinked with TEPA at 1 eq. (solid line), 0.5 eq. (dashed line), and 0.15 eq. (dotted line).

Figure 4. Release kinetics as a function of TEPA crosslinking equivalents of 0.15 eq. (●), 0.5 eq. (■), and 1 eq. (▲). Polysaccharide molecular weights were 1k MD-AMC (solid lines) and 20k Dex-AMC (dashed lines). Error bars for all data points represent standard deviation of triplicate formulations. PS Block Molecular Weight Impacts Release Rates for Small Biologics Increasing PS block molecular weight from 5k to 52k resulted in 3-4 times slower extraction kinetics for the 3k MD-AMC. The reduction was less for the 1k MD-AMC at just 1.3-fold. There was no effect for the 20k Dex-AMC at the two molecular weights tested (5k and 15k). The data for 3k MD-AMC and 20k Dex-AMC are shown in Figure 5 below. The 3k MD-AMC data also indicate a diminishing impact above a 15k PS block. That is, the reduction in rate between 5k PS and 15k PS was much more pronounced than that between 15k and 52k. 288 Ilies; Control of Amphiphile Self-Assembling at the Molecular Level: Supra-Molecular Assemblies with Tuned ... ACS Symposium Series; American Chemical Society: Washington, DC, 2017.


One possible explanation for the reduced release observed with larger PS blocks is greater steric stabilization against NP adsorption to the liquid-liquid interface. NP adsorption would likely result in rapid dissolution of the polysaccharide core into the brine phase as the PS corona is displaced and the PAA core interacts favorably with the aqueous phase. In this picture, the release rate is dominated by coalescence of NPs at the oil/water interface. Inadequate steric stabilization by the polystyrene corona would lead to coalescence and rapid release of encapsulated material at the interface. This mechanism might explain the 3k MD-AMC data. However, the 20k Dex-AMC release profiles show no such PS molecular weight dependence.

Figure 5. Release kinetics for PS blocks of size 5k (▲), 15k (■), and 52k (●) with polysaccharide molecular weights 3k MD-AMC (solid line) and 20k Dex-AMC (dashed line). Error bars for all data points represent standard deviation of triplicate formulations. Alternatively, a mechanism where the PS corona provides a resistance to biologic diffusion could explain the behavior seen in both cases. We can draw analogy from studies on free polymer penetration through existing polymer brushes, which have found greater penetration is possible with shorter brush lengths for a given free chain molecular weight (here, the polysaccharide) (21). Further, studies have identified the importance of the radius of gyration of the free chain in determining penetrability (in addition to other system parameters) (22). In those experiments, larger diffusing chains showed essentially no penetrability into the brush. Release via this proposed mechanism is not the same as brush penetration, but the same steric barrier from the confined chains in the corona controls the system behavior. There is no detectable PS effect for the 20k Dex-AMC because of its relatively large radius of gyration compared to the brush geometry. It should be noted that the changes in polysaccharide loading are a confounding factor which undoubtedly contributed to the profiles reported here. Additional studies in the future will look at this parameter. The important conclusion is that a larger PS block is beneficial for improving EE in the microparticle process. 289 Ilies; Control of Amphiphile Self-Assembling at the Molecular Level: Supra-Molecular Assemblies with Tuned ... ACS Symposium Series; American Chemical Society: Washington, DC, 2017.


Biologic Charge per Mass Impacts Release Rates The impact of the molecular weight of the biologic has been discussed tangentially within the previous sections. In increasing molecular weight from 3k to 20k, a significant drop in release kinetics was observed, as shown in Figure 5. However, the behavior of 1k MD-AMC and 3k MD-AMC did not fit within this trend. Release kinetics were slower for the 1k polysaccharide than for the 3k polysaccharide (9.6% and 24.6% released, respectively, after 40 hours with a 5k PS block). This relative order was true for all PS block sizes tested except for 52k PS, where the release rates were indistinguishable. The amine fluorophore linkage found in MD-AMC holds a positive charge in the inverted NP core and it is well known that a polyacid hydrogel can immobilize cationic molecules (23, 24). Since Scheme 1 ensured a single fluorophore per chain, the 1k MD-AMC had a greater positive charge per mass than the 3k MDAMC. We hypothesized that neutral biologics would restore the expected release order for the 1k and 3k model biologics. The large molecular weight jump between 3k MD-AMC and 20k MD-AMC meant that true size effects could account for the differences described above. In order to study whether charge per mass impacted release kinetics, we produced MD-amide-AMC via Scheme 2. This alternative model biologic contained no charged groups under the PAA core conditions as a result of the amide linkage to the fluorophore.

Figure 6. Release kinetics as a function of positive (●) or neutral (▲) biologic charge and molecular weights of 3k MD (solids lines) or 1k MD (dashed lines) from inverted NPs with a 5k PS corona. Error bars for all data points represent standard deviation of triplicate formulations. Figure 6 presents the release kinetics for 1k and 3k polysaccharides with either positive or neutral net charge. The 1k model biologic showed a significant increase in release rate from the positive to the neutral form, as expected. This would indicate decreased charge interactions with free acid groups on the PAA Further, the neutral 1k and 3k MD-amide-AMC exhibited nearly identical 290 Ilies; Control of Amphiphile Self-Assembling at the Molecular Level: Supra-Molecular Assemblies with Tuned ... ACS Symposium Series; American Chemical Society: Washington, DC, 2017.

behavior. This may indicate that the experimental technique is not sufficiently precise to distinguish between these molecular weights. However, the 3k polysaccharide exhibited a slightly slower release profile in the neutral form than the positive form. The decrease in release kinetics may be due to differences in some physical property as a result of the alternative synthetic scheme. For example, the stochastic nature of fluorophore linkage formation may result in chains with more than one fluorophore, reducing the driving force for dissolution into the brine phase. Neutral model biologics modified only via the reducing end of the chain would enable direct comparisons with less uncertainty.


Resistances To Release Describe the Extraction Process Through this work, we have sought to understand how inverted NP formulations will impact diffusion of the biologic (and consequently the microparticle EE) by identifying release rate differences. In the previous sections, we have begun constructing an explanation for the observed release profiles in terms of resistances to biologic diffusion. This is shown schematically in Figure 7. Resistance R1 is the PAA hydrogel. Our data suggest this resistance term is important in two cases: (1) for large biologics whose size likely approaches the average distance between hydrogel crosslinks, and (2) for small biologics with a more positive charge/mass. For a typical application of iFNP, the physical properties of the biologic will be pre-determined, rendering this a non-adjustable parameter. Resistance R2 is the PS corona. Recall that the corona does provide a steric barrier against absorption to the liquid-liquid interface, but that this physical picture does not fully described the experimental observations. We must also consider the steric barrier to biologic diffusion presented by the corona. This barrier depends strongly on the radius of gyration of the model biologic. Finally, the chloroform phase acts as resistance R3. However, the complete extraction of the control samples to the brine phase within the first thirty minutes indicates that this is a negligible resistance which can be ignored. To describe diffusion from a reservoir through a barrier, we would expect first order kinetics. However, this model does not provide a good fit for the data, with systematic errors observed in the linearized data. For release from a spherical monolith, we expect similar behavior (25). A good fit for the data is provided by a t1/2 dependence, which describes diffusion through a thin film. Figure 8 shows such a fit applied to 20k Dex-AMC release data at different crosslinking densities (R2 > 0.97 for both data sets). One should note that the different slopes provide a clear metric of the impact of higher TEPA crosslinking on release kinetics for large biologics. A t1/2 dependence would also be expected for a mechanism of NP absorption to the interface followed by rapid release of core contents (26). However, as discussed above, this mechanism does not completely explain the data set. Care must be taken not to inappropriately draw conclusions regarding the release mechanism from a fitted model as the superposition of different minor effects can drastically change the apparent release kinetics. 291 Ilies; Control of Amphiphile Self-Assembling at the Molecular Level: Supra-Molecular Assemblies with Tuned ... ACS Symposium Series; American Chemical Society: Washington, DC, 2017.


Figure 7. Resistances to diffusion of core material in the extraction assay. R1 is the PAA hydrogel structure. R2 is the PS brush. R3 is the organic solvent surrounding an individual inverted NP.

Figure 8. Release kinetics for 20k Dex-AMC fit to a diffusion-controlled release model (m is the slope of the fit line) with TEPA crosslinking equivalents of 0.5 eq. (■) and 1 eq. (▲). Error bars for all data points represent standard deviation of triplicate formulations.



Application to Microparticle Processing The extraction studies reported here do not provide direct insight into the anticipated release profiles from microparticles produced using inverted NPs. However, we believe they provide insight into the mechanisms that determine EE during processing. These studies have demonstrated a primary benefit of replacing pores with inverted NPs: the added protection afforded by the BCP. Further, the experiments have identified several tunable parameters which influence the rate of biologic loss during exposure to an aqueous phase. We applied some of these learnings to evaluate the encapsulation efficiency in a model non-degradable microparticle. We used polystyrene as the scaffold polymer to match the inverted NP corona. The effect of the biologic molecular weight and the external aqueous phase composition were studied first. We found that encapsulation efficiencies were universally very high, with all tested formulations greater than 80%. The addition of brine to the external phase resulted in the expected reduction in losses for both formulations, with a stronger effect observed for the 20k Dex-AMC, as shown in Figure 9. This is likely due to the higher core water content of that formulation. With brine present, there was no discernable difference in EE between the 3k MD-AMC and the 20k Dex-AMC formulations. This may reflect that the extraction assay is more sensitive to differences between the formulations or that some other mechanism is dominant in determining losses. The observation of lower EE for the 20k Dex-AMC without brine suggests the latter is the case.

Figure 9. Encapsulation efficiency of the microparticle process with 3k MD-AMC (black) and 20k Dex-AMC (grey) inverted NPs with a 5k PS corona. The groupings indicate an external aqueous phase with or without 7.5 wt% NaCl. The overall process encapsulation efficiency (accounting for losses during the brine extraction to remove DMSO and MP formation) was 91% ± 1% for both 3k MD-AMC and 20k Dex-AMC when using a brine external phase. This microparticle formulation produces particles which are 50% inverted NPs (corresponding to a target 25% biologic loading). Loadings and encapsulation 293 Ilies; Control of Amphiphile Self-Assembling at the Molecular Level: Supra-Molecular Assemblies with Tuned ... ACS Symposium Series; American Chemical Society: Washington, DC, 2017.

efficiencies of this magnitude represent significant improvements over typical values reported in the literature (27). Currently, we are transitioning to a biodegradable BCP system for further studies. We will evaluate the impact of the hydrophobic block size as well as biologic characteristics on EE. The degradable system will also allow for release studies to be carried out. In our on-going work, we are evaluating protein processing with iFNP to understand the impact on secondary structure and activity, which were not able to be evaluated in the present work.


Conclusion We have described a novel model biologic system consisting of fluorescently-labeled polysaccharides, which allow for inexpensive evaluation of iFNP formulations. With this system, key characteristics of the biologics can readily be manipulated while holding other variables constant. With an extraction protocol, we have determined that a number of formulation parameters impact processing losses, such as brine content of the aqueous phase. Based on the findings, we demonstrated encapsulation efficiencies in excess of 90% for microparticle formulations that contained a target of 25% biologic loading. Potential additional variables to assess are corona chain length, extent of crosslinking, and charge for smaller biologics. This work has demonstrated one of the benefits of inverted NPs for microparticle production – the reduction of process losses through the BCP barrier to diffusion. This enables scalable biologic formulations that are economical, with the potential for new treatment options for patients.

Acknowledgments We would like to thank Istvan Pelczer and Ken Conover of the Princeton University NMR Facility (Department of Chemistry) for their assistance in characterization of the reaction products. CEM would like to thank Robert Pagels for helpful conversations about this work. This work was supported by a grant from the Princeton Old Guard Fund and the Princeton Innovation Fund. Support is acknowledged from Optimeos Life Sciences, Inc.

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Inverse Flash NanoPrecipitation for Biologics Encapsulation

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