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Preparation and Characterization of Biomimetic #-Lens Crystallins Using Single-Chain Polymeric Nanoparticles Jue Liang, Jessica J. Struckhoff, Paul D Hamilton, and Nathan Ravi Langmuir, Just Accepted Manuscript • DOI: 10.1021/acs.langmuir.7b01290 • Publication Date (Web): 28 Jun 2017 Downloaded from http://pubs.acs.org on July 3, 2017

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Preparation and Characterization of Biomimetic β-Lens Crystallins Using Single-Chain Polymeric Nanoparticles Jue Liang1, Jessica J. Struckhoff1, Paul D. Hamilton1,2, and Nathan Ravi1,2,3* 1

Department of Ophthalmology and Visual Sciences, Washington University School of

Medicine, Saint Louis, MO, 63110 2

Medical Research, VA Medical Center, Saint Louis, MO, 63106

3

Department of Energy, Environmental and Chemical Engineering, Washington

University, Saint Louis, MO, 63110

Correspondence to: Nathan Ravi; Mailing address: Department of Ophthalmology and Visual Sciences, Washington University in St. Louis; 660, South Euclid, Campus box 8096, St. Louis 63110, USA. Tel.: +1 314-747-4458; fax: +1 314 -747-5073 E-mail address: [email protected] KEY WORDS: lens crystallin, single chain nanoparticles, protein mimetic, ECIS

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Abstract Presbyopia, the inability to focus at arm’s length, and cataracts that cloud vision are associated primarily with changes in the mechanical and optical properties of the lens crystallins. The optical properties, particularly the refractive index, of the human lens originate from the cytoplasm of the lens fiber, which contains a highly concentrated solution (~40%) of globular proteins referred to as α, β, and γ crystallins, of which β is the most abundant. In this study, we focus on the synthesis and characterization of a βcrystallin biomimetic in an effort to understand and develop treatments for presbyopia and cataract. Polyacrylamide was used as a protein analog. The side chains were endowed with aromatic and acidic functionality. Acrylic acid was incorporated into the copolymer and crosslinked with diamines to form nanoparticles. The composition and crosslinking condition of the biomimetic copolymers were optimized to match the hydrodynamic radius (Rh), refractive index, size, density, and intrinsic and dynamic viscosities with those of βhigh lens crystallins. The refractive indices and densities of the nanoparticles’ dispersion at different concentrations matched that of βhigh lens crystallins, and the viscosity of the nanoparticles approached that of βhigh lens crystallins. The biocompatibility findings for primary porcine retinal pigment epithelial cells (ppRPE) and porcine lens epithelial (pLE) cells showed both cell types tolerated up to 30 mg/mL of nanoparticles. These materials have the potential for use as replacing the crystallins in developing an accommodating intra-ocular lens nanocomposite hydrogel that closely replicates the natural autofocusing ability of the original.

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Introduction Proteins differ from synthetic polymers in that their polyamide backbones are degradable and they have a single unique molecular weight because of carefully controlled enzymatic condensation reactions utilizing a limited variety of amino acids. Proteins have a number of pendant functional groups in addition to amino and carboxyl terminal groups. These pendant functional groups endow each protein with signature properties. The side groups are also susceptible to post-translational modification that could potentially alter the physiological properties. Proteins typically have a globular or fibrillar quaternary structure that is a function of their amino acid sequence. Synthetic polymers, on the other hand, can be made from a variety of monomers. They can be biodegradable or non-biodegradable, linear, branched, star-shaped, comb- or brushshaped, dendritic, crosslinked, etc. Except for the dentritic motif, they usually exhibit a molecular weight distribution. The human lens is an auto-focus component of the visual system that enables humans to focus clearly at varying distances. The process that enables the eye to focus from a distant object to a near object is referred to as accommodation.1, 2, 3 The reverse, when focusing from near to distant, is disaccommodation. Accommodation and disaccommodation processes are imperceptible and appear to occur almost instantaneously. With advancing age, the amplitude as well as the speed of accommodation is decreased, significantly leading to a condition called presbyopia, which is treated inadequately with bifocals. At around sixty years of age, the lens begins to scatter light, which is clinically referred to as a cataract. The pathophysiology of presbyopia and cataract formation is associated with changes in the lens volume from its continued growth, changes in the lens

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viscoelasticity, and changes in the chemical and physical state of the lens crystallins.3, 4, 5, 6, 7, 8

Cataract occurs when the quality of vision worsens due to scattering of aggregated

crystallins. Treatment is surgical. A small opening is made in the lens capsular bag and the contents are broken up with ultrasound and aspirated. The contents of the evacuated lens capsular bag are replaced with an intra-ocular lens that provides minimal or negligible accommodation. Consequently, there exists an opportunity during surgery to fill the evacuated bag with a soft nano-composite hydrogel that mimics the natural lens in its optical, physical, and mechanical properties. Our interest in synthetic proteo-mimetics stems from our continued effort to understand the molecular origins of the human lens, particularly its physical, mechanical, and optical properties. Our previous attempt to develop a single component hydrogel was not successful; if we matched the mechanical properties, the refractive index was too low, and vice versa. After examining the contents of the lens capsular bag with respect to the molecular origins of their elastic modulus, viscosity, density, optical properties, and relaxation time constants, we have discovered that Nature uses a two-component system. In this system, the cytoskeletal proteins provide the mechanical properties, including the elastic and rapid relaxation time constants of the lens, and globular proteins, called lens crystallins, provide the optical and viscous properties, including the slower relaxation time constants than the cytoskeletal proteins.9,10 The lens crystallins can be fractionated by size and charge into α (high and low), β (high and low), and γ crystallins. Of the lens crystallins, β high is the most abundant. It has about 3% thiol/disulfide groups, a pI close to neutral pH, a molecular weight of ~140 kD, and a diameter of around 10 nm. It is made up of subunits and is often found as tetramer.

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We used derivatives of acrylamide as monomers to form a non-degradable carbon-carbon backbone with pendant amides. To form polymeric nanoparticles, we previously utilized disulfide chemistry.10 In this study, we used the formation of amide bonds to make intramolecular crosslinks which gave rise to nanoparticles. An aromatic component was incorporated to make the nanoparticles more compact and to enhance the refractive index. Here we report on the preparation and characterization of nanoparticles that closely resemble the βhigh lens crystallin in refractive index, size, density, and viscosity, and that have acceptable biocompatibility. Experimental Materials Acrylamide (AM), acrylic acid (AA), N-phenylacrylamide (NPA), N,N,N’,N’tetramethylethylene diamine (TEMED), ammonium persulfate (APS), 2-chloro-4,6dimethoxy-1,3,5-triazine (CDMT), acetonitrile, 4-methylmorpholine (NMM), hexamethylenediamine (HMDA) and sodium azide were purchased from Sigma Aldrich. Preparation of copolymer A characteristic procedure for synthesizing poly(AM-NPA-AA) copolymer with a molar ratio of 85.5:7.5:7 (AM:NPA:AA) is described. The polymerization was conducted with 5 wt% of monomer in 25 v% ethanol (25:75 ethanol:water). A mixed solvent was used because of the limited solubility of NPA in water. Desired amounts of AM (3.95 g, 55.6 mmole), NPA (0.718 g, 4.88 mmole), AA (313 µL, 329 mg, 4.60 mmole), and TEMED (1.00 mL, 776 mg, 6.69 mmole) were dissolved in 25 mL of 25 v% ethanol. After the complete dissolution of the monomers, 75 mL of water was added. Nitrogen was bubbled through the solution for 30 min to remove any dissolved oxygen. APS (0.20 g,

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0.88 mmole) was dissolved in 1 mL of water and added to the mixture to initiate the freeradical polymerization. The mixture was bubbled with nitrogen for another 5 min, and the reaction was allowed to proceed for 15 h at 22 ̊C. The copolymer was dialyzed (MWCO =12,000 -14,000) against DI water (4 L×5) for 5 days to remove any unreacted monomers and other small molecules. Preparation of nanoparticles Nanoparticles were prepared from poly(AM-NPA-AA) copolymer as follows: By using the poly(AM-NPA-AA) copolymer (AM:NPA:AA=85.5:7.5:7), one liter of 0.1 % w/v copolymer solution was prepared and further sonicated for 5 min. A 5% w/v CDMT/acetonitrile solution (32 mL, 1.6 g, 9.1 mmol), NMM (1.50 mL, 1.38 g, 13.7 mmol) and hexamethylenediamine (58 mg, 0.5 mmol) were added sequentially. The solution was stirred for 40 h and concentrated to ~200 mL using an Amicon DC2 hollow fiber concentrator (Millipore Corporate, MWCO=10,000). The small molecules were removed by dialysis (MWCO=12,000-14,000) against DI water (8 L×5) for 5 days. This solution was further concentrated using an Amicon centriprep concentrator (Millipore Corporate, MWCO =10,000) to a higher concentration of 30 wt%. Characterization of compositions by NMR and titration The contents of NPA in the copolymers were measured by 1H NMR. 1H NMR spectra were obtained on a Varian Unity Inova 500 MHz (Palo Alto, CA). Copolymers were dissolved in D2O (20 mg/mL) and each sample was scanned 16 times at 25 ̊C. The copolymers and the nanoparticles were titrated with a 0.1 N NaOH aqueous solution, using a pH meter to measure the contents of AA and amines. An aliquot of the copolymer or nanoparticle was dispersed in 10 mL of DI water. A 0.1 N NaOH aqueous

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solution was added to the mixture in a defined volume, and the pH was tested with a pH meter after each addition. Characterization by gel permeation chromatography The molecular weights, intrinsic viscosities and hydrodynamic radii of the copolymers and the corresponding nanoparticles were determined using a Viscotek GPC system equipped with viscosity, static light scattering, and refractive index detectors. The pump was linked with the stationary phase consisting of a dual column of G6000PWXL, G4000PWXL, connected in series. The mobile phase was a 20-mM Bis-Tris buffer (pH 6.0, 0.1% sodium azide), and the flow rate was 0.8 mL/min. The measurements were conducted at 37 ̊C. Rheological measurements The dynamic viscoelastic characteristics of 6 wt% solutions of the copolymers, nanoparticles, hydrogels, and β-high crystallin were compared using a Vilastic-3 Rheometer (Vilastic Scientific, Austin, TX). Each sample was measured at a frequency of 2 Hz at 22 ̊C in a cylindrical tube with a radius of 0.04953 cm and length of 6.278 cm. Measurements were performed over a range of shear rates from 0.2 /sec to 50/sec. For each sample, measurements were performed in duplicate. In all of the samples, the concentrations of dibasic buffer and sodium azide were maintained constant at 10 mM and 0.1% respectively. Determination of refractive index The refractive index values at different concentrations (5, 10, 15, 20, 25 and 30 wt%) for the nanoparticles and β-high crystallin were determined using an Abbe refractometer (ATAGO’s Abbe refractometer 1T, Kirkland, WA) at 25 ̊C.

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Biocompatibility test The extraction and passaging procedures for primary porcine retinal epithelial (ppRPE) cells were established as previously described.11 ppRPE cells were cultured for 17-24 days after extractions and used at passage 1. Primary porcine lens epithelial (ppLE) cells were used at passages 6 and 7. For the presented experiments, both cell types were grown in tissue culture media (CDF-12) comprised of DMEM/F-12, 10% FCS, supplemented with MEM nonessential amino acids, sodium pyruvate, and L-glutamine, 100 U/mL penicillin, 1 mg/mL streptomycin, and 2.75 ug/mL amphotericin. In vitro biocompatibility was analyzed using electric cell-substrate impedance sensing technology (ECIS Ztheta, Applied BioPhysics, Troy, NY). The resistance and impedance measurements were recorded over 168 hours for cells distributed in a tissue culture well. 96-well 10idf ECIS arrays were prepared according to the manufacturer’s instructions. Arrays were pretreated with 10 mM sterile cysteine in water that provided a coating for the gold electrodes via the interaction of the –SH groups in cysteine with the gold surface, which increased the reproducibility of cell attachment and spreading. Cell culture media was added to the wells for protein absorption to the gold electrode surface. Resistance measurements and complex impedance measurements gave identical curves at 4,000 Hz. ppRPE cells were plated at 12,000 cells/well, and ppLE cells were plated at 7,000 cells/well. These cell densities were chosen to show how the cells reacted to the nanoparticles as they grew toward a confluent monolayer. The cells were grown for 24 hours in the culture plate to allow the cells to adhere, the media was then removed, and the nanoparticles in cell culture media were applied directly to the cells. Preparation of βhigh crystallin solution

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Porcine eyes were obtained from a local abattoir (Weyhaupt, Belleville, IL) and lenses were dissected out. Decapsulated lenses were placed in a buffer, (50 mM Tris, 50 mM NaCl, 1 mM EDTA, 1 mM DTT and 0.1% sodium azide), homogenized, and centrifuged at 12,000 rpm for 30 min. Approximately 1.2 g of the soluble fraction was loaded on a 5 X 90 cm Sephacryl S-300 Column (Pharmacia) and fractions were collected every 10 minutes at a flow rate of 1.3 mL/min. Absorbance was measured at 280 nm using an ISCO model UA-5 monitor. Fractions were pooled into their respective peaks of α, β high, β low, and γ crystallins. The β high was concentrated using an Amicon DC2 hollow fiber concentrator (Millipore Corporate, MWCO=10,000). It was further concentrated using an Amicon centriprep concentrator (Millipore Corporate, MWCO =10,000) to a higher concentration of about 25 wt%, during which the buffer was exchanged with 10 mM dibasic phosphate and 0.1% sodium azide. The final β high crystallin solution was stored at 4 ̊C.

Results and Discussion Optimization of the composition of the poly(AM-NPA-AA) copolymer In this study, we focus on developing materials that can make up the optical part of a nature-mimicking two-component lens system that will enable us to probe the accommodation process of the lens. We have studied this process using a whole lens in a robotic lens stretcher in real time.12 Challenges in obtaining a working material include matching the refractive index, density, transparency, and viscoelastic response of the synthetic material to that of the natural lens. Accommodation occurs very rapidly, and hence the materials used must respond accordingly. We measured the relaxation time

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constants of the natural porcine lens (t1 34.5 ms, t2=310 ms, t3=12,400 ms). The force magnitudes for various force-decay processes in accommodation were calculated for three parallel Maxwell elements in parallel with an elastic solid, dependent upon the elastic and viscous properties of the lens, the latter originating from the lens crystallins. The lens cytoplasm is composed of approximately 60 wt% of water, with the balance primarily composed of proteins. The globular cytoplasmic proteins referred to as crystallins constitute over 95% of the proteins. The optical properties of the lens originate from the highly concentrated solutions of crystallin proteins (~40 wt%). The unfractionated lens crystallin solution exhibits liquid-like behavior with a high refractive index (1.4), low density (1.09 g/mL), and low viscosity (~ 1.45x10-3 Pa·s). These observations shaped the genesis of a biomimetic nanocomposite with the appropriate properties.13 In addition, the envisioned nanoparticles should have hydrodynamic sizes that are close to that of crystallin proteins (Rh of β high crystallin=~5 nm).9 Nanoparticles with appropriate sizes would avoid light scattering or diffusion from the lens capsule.14 We have previously reported the utilization of polymeric nanoparticles and organo-silica nanoparticles as substitutes for lens crystallin.10 However, they variously had a low refractive index (1.358) (polymeric nanoparticles), a small size (Rh=~1.5 nm) (organo-silica nanoparticles), or transitioned from a nanoparticle to a gel over time due to the reversible characteristics of disulfide bonds. The high refractive index of lens proteins is attributed to the amide bonds in their backbones. In this study, we used polyacrylamide, which has amide bonds in the side chain in each repeating unit, as the major component of the nanoparticles, due to its high refractive index and high solubility in water. In addition, we incorporated an aromatic

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monomer, NPA, to make the nanoparticles more compact by hydrophobic interaction and to increase the refractive index. An ionic monomer, AA, was introduced to the copolymer to provide sites for crosslinking by forming amides with diamines. In this work, intra-chain crosslinking is desired for forming single-chain nanoparticles with a compact structure. However, besides the individually desirable properties of the three monomers, each has its own disadvantages for making crystallin-mimetic nanoparticles. The polyAM backbone is very hydrophilic, and thus will not form a compact structure in water. The hydrophobic NPA makes the nanoparticles more likely to aggregate, and thus to lose their transparency. The ionic AA makes the backbone of the copolymer more rigid by electrostatic repulsion, and thus it becomes less compact and less likely to form intra-chain crosslinks. Addition of the AA component also reduces the refractive index of the composite. However, taken all around, the disadvantages of each component can be counterbalanced by the advantages of the other two components. Therefore, to carefully optimize the composition of the poly(AM-NPA-AA) copolymer, we synthesized and compared a series of copolymers with different compositions.

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Figure 1. A characteristic 1H NMR spectrum of poly(AM-NPA-AA) copolymer containing 7.5 mol% of NPA and 7 mol% of AA. The copolymers were characterized with 1H NMR (Figure 1). The contents of NPA were calculated from the integration ratios of the aromatic protons at ~7.0-7.5 ppm to the backbone protons at 1.0-2.5 ppm. The integration of the CH2 and CH in the backbone was set as 300, standing for 100 repeating units for easy calculation. Therefore, the molar percentage of NPA in the copolymer could be calculated by dividing the integration at 7.0-7.5 ppm by 5, since there are 5 protons attached to one aromatic ring. Because the resonance of AA in the 1H NMR spectrum was not distinguishable from the other components, the AA contents were measured by acid-base titration. The results from 1H NMR and titration indicated that NPA and AA were incorporated into the copolymers at the feed ratio (Table 1). The molecular weights and intrinsic viscosities of the copolymers were measured by GPC (Table 1). Generally, polymers that have more compact structures tend to have lower intrinsic viscosities.15 The intrinsic viscosities of 12


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our poly(AM-NPA-AA) copolymers increased with an increasing molar ratio of AA (shown by comparing entries 3, 4 and 5, or entries 6, 7 and 8). This resulted from the electrostatic repulsion of the negatively charged acrylate repeating units, which made the chain more likely to expand and resist forming a compact structure. On the other hand, the intrinsic viscosities decreased with increasing molar ratios of NPA (shown by comparing entries 1, 5 and 7 or entries 2, 4 and 6). This result was due to the aggregation of hydrophobic repeating units, which makes the polymer chain more compact. In any event, copolymers containing 10 mol% of NPA formed a significant amount of intermolecular aggregates that made the solution hazy, so they were eliminated from further study. Table 1. Characterization of poly(AM-NPA-AA) copolymers with different compositions. Entry

NPA %

AA %

1 2 3 4 5 6 7 8

3 5 7.5 7.5 7.5 10 10 10

7 5 3 5 7 5 7 10

Mn (kDa) 52 49 56 53 56 58 52 54

Mw (kDa) 102 72 82 89 84 88 75 83

IV (dL/g) 0.73 0.57 0.51 0.5 0.59 0.45 0.48 0.55

PDI

Yield

1.96 1.49 1.48 1.69 1.50 1.53 1.46 1.54

88.2 90.9 77.5 69.5 81.5 65.8 67.7 69.1

We synthesized nanoparticles by crosslinking the poly(AM-NPA-AA) copolymers with HMDA.

Because of its excellent efficiency and low cost, a triazine-based coupling

method was utilized for crosslinking. The triazine-based coupling method has been previously used for synthesizing or modifying peptides,16, 17 nucleic acids18, 19, 20 and

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polysaccharides.21, 22 In this work, the AA repeating units reacted with the diamine and formed amide bonds in the presence of CDMT and NMM as the catalyst. A series of copolymers with different compositions were crosslinked by employing a constant molar ratio of CDMT (CDMT:AA=10:1), NMM (NMM:AA=15:1) and HMDA (HMDA:AA=0.55:1) (Table 2). The crosslinking reactions were conducted at a very diluted concentration (0.1 wt%) to form single-molecule nanoparticles, which would have lower intrinsic viscosities than multi-molecule nanoparticles. The increase in molecular weights after crosslink was only ~10%, which suggests that intra-chain crosslinking predominated over inter-chain crosslinking. Thus, we were able to synthesize singlemolecule nanoparticles. Nanoparticles made from copolymers with 7.5 mol% of NPA had lower viscosities than those made from copolymers with 3 or 5 mol% of NPA (as shown by comparing entries 1 and 5, or entries 2 and 4), which was due to the hydrophobic interaction discussed earlier. In addition, the intrinsic viscosities of the nanoparticles were significantly reduced from the uncrosslinked copolymers, which confirmed the formation of more compact structures. Nanoparticles made from copolymers with more AA had less viscosity (seen by comparing entries 3, 4 and 5), which was due to their higher degrees of intra-chain crosslinking. In conclusion, the copolymers with 7.5 mol% of NPA formed the most compact nanoparticles while being hydrophilic enough to avoid inter-molecular aggregation. Moreover, copolymers with 7 mol% of AA provided the greatest reduction of viscosity while maintaining a refractive index as high as that of βhigh crystallin (data shown in the next section). Therefore, the poly(AM-NPA-AA) copolymer with 7.5 mol% of NPA and

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7 mol% of AA was determined to have the optimum composition for making crystallin proteo-mimetic nanoparticles. Table 2. Characterization of nanoparticles with different compositions. NPA

AA

Mn

Mw

Rh

IV

IV

%

%

(kDa)

(kDa)

(nm)

(dL/g)

reduction

1

3

7

47

105

8.3

0.42

42%

2

5

5

53

99

7.9

0.38

33%

3

7.5

3

66

100

8.3

0.41

20%

4

7.5

5

58

85

7.3

0.32

36%

5

7.5

7

49

69

6.1

0.25

58%

Entry

Optimization of the formulation for crosslinking the copolymer We also optimized the formulation for crosslinking the copolymer by varying the molar ratio of CDMT and NMM to AA. The two amino groups in HMDA were expected to react with the AA repeating units to form amide crosslink sites. However, it is also possible that only one of the two amino groups in HMDA reacted with AA, and this would introduce free amines into the nanoparticles and reduce the degree of crosslinking. Therefore, the nanoparticles and uncrosslinked copolymers were titrated to measure the contents of unreacted AA and free amino groups. Figure 2 shows characteristic titration curves (Top) of nanoparticles, uncrosslinked copolymer and water, as well as their corresponding differential pH curves (bottom). The inflection points in the titration curves indicate the buffer ranges of AA (pH=3.7-7 ) and amines (pH=7-9), respectively. Comparing the titration curves of nanoparticles and uncrosslinked copolymer reveals

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partial consumption of the AA and the formation of free amino groups in the nanoparticles. Thus, the degree of crosslink could be calculated by subtracting the unreacted AA and the free amines in the nanoparticles from the total amount of AA in the uncrosslinked copolymer. By varying the molar ratio of CDMT and NMM, we were able to tune the degree of crosslink, the hydrodynamic size, and the intrinsic viscosity of the nanoparticles (Table 3). By using 10 moles of CDMT and 15 moles of NMM (compared to AA), we converted 93% of the AA to amide, crosslinked ~90% of the AA, reduced the intrinsic viscosity by 58%, and reduced the hydrodynamic size from 8.8 nm to 6.1 nm. Further increasing the amount of NMM did not make a significant difference. In addition, the nanoparticles (Rh=6.1 nm) were slightly larger than the βhigh crystallin protein (Rh= ~5 nm), which could beneficially prevent diffusion of the nanoparticles from the pores of the lens capsule.

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Figure 2. Characteristic titration curves and dpH curves of water, poly(AM-NPA-AA) copolymer, and nanoparticles synthesized with CDMT:NMM:AA=1:1:1.

Table 3. Characterization of nanoparticles made with different crosslinking formulations. .

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CDMT: NMM:

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Unreacted

Free

Crosslink

Rh

IV

IV

Entry AA

AA

AA%

Amines%

%

(nm)

(dL/g)

reduction

Control

0

0

100

0

0

8.8

0.59

0

1

1

1

47.4

18.4

34.2

8.3

0.49

17%

2

3

5

30.3

7.1

62.6

8.0

0.41

31%

3

6

10

14.7

9.2

76.1

6.8

0.31

47%

4

10

15

7.0

3.5

89.5

6.1

0.25

58%

5

10

20

7.1

4.1

88.8

6.3

0.26

56%

Comparison of refractive indices βhigh crystallin protein has a high refractive index, which is critical for the optical property of the natural lens. Previous research has attempted to make polymeric nanoparticles for mimicking crystallin proteins.10 However, the refractive index of those nanoparticles was much lower than that of βhigh crystallin. In this research, the choice of monomers and their ratio not only make the nanoparticles compact, but also endow them with a high refractive index. The refractive index of our poly(AM-NPA-AA)-based nanoparticles matches well with that of βhigh crystallin protein, as shown in Figure 3.

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Figure 3. Refractive index values of βhigh lens crystallin and our produced nanoparticles (containing 85.5% of AM, 7.5% of NPA and 7% of AA) at different concentrations.

Comparison of densities Another important feature of the natural lens is its low density (1.09 g/mL), even with a very high concentration of crystallin proteins (40 wt%). When silica nanoparticles, which also have a compact structure and high refractive index, were tested in our lens composites, their greater density increased the density of the lens composite to a level significantly above the physiological density of the natural lens. This increase caused problems with the lens capsule not being able to maintain its symmetry and stretching of the ciliary body. These problems showed the necessity of matching the physiological density of the lens materials (~ 1.09 g/mL). The density of an aqueous solution of our polymeric nanoparticles was tested and compared to the natural lens and a suspension of 12-nm silica nanoparticles (LUDOX® HS30 and HS 40). As shown in Figure 4, the 19


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density of the polymer-based nanoparticles solution was close to that of pure water, even at 30 wt% (1.05 g/mL). In contrast, the density of the silica nanoparticle solution at 30 wt% was significantly higher.

Figure 4. Densities of 12 nm silica nanoparticles, and 12 nm polymeric nanoparticles (containing 85.5% of AM, 7.5% of NPA and 7% of AA) at different concentrations. Density of the lens is used as a reference point. Comparison of dynamic viscosity The viscoelastic characteristics of the uncrosslinked copolymer, nanoparticles, and βhigh crystallin were compared at the same concentration (6 wt%) (Figure 5). The viscosities increased in the order of βhigh crystallin < nanoparticles < copolymer. The βhigh crystallin showed the lowest viscosity of 1.45x10-3 Pa·s due to the highly compact structure of the protein molecule. The viscosity of the nanoparticles was measured as 3.0x10-3 Pa·s, which was about 50% less than that of the corresponding polymer solution (5.8x10-3 Pa·s). This lower viscosity showed the enhanced compactness of the nanoparticles when compared with that of the copolymer, which was achieved by the

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intramolecular crosslinking and confirmed the GPC data mentioned above. A more precise control of the narrow molecular weight distribution of the copolymer and the balance charge and hydrophobicity may lead to nanoparticles with even lower viscosity. Our future work is progressing in this direction.

Figure 5. Viscosity of β high lens crystallin, the copolymer, and nanoparticles (containing 85.5% of AM, 7.5% of NPA and 7% of AA) at 6 wt%. All of the samples contained 10mM phosphate and 0.1% sodium azide at pH 7.5.

Biocompatibility tests We selected nanoparticles formed by copolymer containing 85.5% of AM, 7.5% of NPA and 7% of AA as the optimal formulation for the biocompatibility experiments. ppRPE cells were tested to show how the retinal barrier might be affected should nanoparticles escape the lens capsule. Similarly, pLE cells were tested to show how the lens epithelial layer might be affected should the epithelial cells grow inside the capsule, which is commonly seen with cataract surgery. Ideally, the nanoparticles should be contact

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cytotoxic to remnant epithelial cells in the capsular bag, so posterior capsular opacification does not occur. Electric cell-substrate impedance sensing (ECIS) technology was used to determine how the cells fared while directly exposed to nanoparticles for six days. ECIS is a non-invasive technique wherein a current is applied, and the resistance, impedance, and capacitance of cells adhered to gold electrodes is recorded. The results show the resistance of each cell type changes as a current of 4,000 Hz is applied. Resistance changes indicate changes in the cells covering the surface of the well. An increase in resistance can be caused by an increase in cell number or a change in cellular morphology. Toxicity is assessed by comparing the changes in the resistance of cells exposed to nanoparticles to changes in resistance for control cells exposed to only cell culture media. This technique can provide continuous detailed, objective, in vitro biocompatibility and cellular responses to nanoparticles. The data presented are representative of the findings from three experiments, with five to eight experimental repeats per condition per experiment. Figure 6 shows the resistance measurements for ppRPE cells over 7 days. When the nanoparticles were added with media change at 24 hours, there was an initial jump in resistance for all conditions, including the control. Cells exposed to nanoparticle concentrations from 0.1 mg/mL to 5 mg/mL showed a continued increase in cellular resistance for 48 hours, which follows the curve of the control cells. However, cells exposed to 1 and 5 mg/mL showed overall lower resistance than control cells for the duration of the experiment. For 15 to 30 mg/mL, the resistance stagnated after nanoparticle addition, and after 48 hours of exposure, the resistance readings started to decline, indicating the cells were not healthy. Cells exposed to 30 mg/mL experienced a

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rapid decline in resistance after 48 hours, giving resistance readings essentially the same as the media controls by 168 hours. The visual findings of the cell morphology (Figures 7) substantiated the ECIS curves for ppRPE. Cells exposed to up to 5 mg/mL nanoparticles appeared similar to control cells in size, shape, and cell numbers. Starting at 15 mg/mL, the cells showed changes in cell organization: Cells appeared larger, with a slight increase in distance between cells. Additionally, there was a steady increase in cytoplasmic vacuoles seen at 15-30 mg/mL exposure. Cells exposed to 15-30 mg/mL likely did not divide further after the addition of nanoparticles. Cell death was apparent for cells exposed to 30 mg/mL for 6 days.

In

all nanoparticle concentrations except 30 mg/mL, the cellular morphology and amount of cytoplasmic vacuoles remained steady from day 3 (data not shown) to day 6. The most likely conclusion is that if the retina encounters our engineered nanoparticle, its effects would be dilute and transient. Our results confirm that in very low concentrations, the nanoparticles have minimal effect. But as the concentration increases, ppRPE experience changes in morphology and the ability to maintain resistance. Even at the highest concentration, it took over five days to completely kill the cells. Thus, the ppRPE cells displayed an acceptable ability to maintain their morphology and resistance over the course of the exposure, with tolerance increasing with decreasing nanoparticle concentration.

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Figure 6. ECIS resistance measurements of ppRPE cells exposed to nanoparticles for 144 hours. Nanoparticles were added at 24 hours and measurements were continuous over 168 hours.

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Figure 7. Light microscope images taken at 10X of ppRPE cells exposed to nanoparticles for 6 days. Figure 8 shows the resistance measurements for pLE cells over 7 days. Cells treated with increasing nanoparticle concentration show a greater resistance than controls. ppLE cells appeared to tolerate the nanoparticles better than ppRPE cells, in that the cells continued to grow and adhere to the plate for 48 hours after exposure to all nanoparticle 25


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concentrations. At that point, cells exposed to 15-30 mg/mL experienced a sustained decrease in resistance until 7 days (the end of the experiment). However, it is only during the period from 5-7 days that the 15-30 mg/mL conditions have resistance values that drop below control values. The resistance did not drastically reduce, as it did for the ppRPE cells. This finding indicates that the cells were still alive, but that they are likely undergoing morphological changes and possibly pulling away from each other, reducing the strength and number of their tight junctions. The indicaton is that nanoparticles would be toxic to the lens epithelial cells at very high concentrations.

Figure 8. ECIS resistance measurements of pLE cells exposed to nanoparticles. Nanoparticles were added at 24 hours and measurements were continuous over 168 hours.

The cell morphology (Figures 9) displays the reaction of ppLE cells to nanoparticle exposure as followed by ECIS curves. Cells exposed to up to 5 mg/mL of nanoparticles appeared similar to control cells in size, shape, and cell number. There was

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a steady increase in cytoplasmic vacuoles seen from 15-30 mg/mL, but the cellular structure appeared comparable to control cells even at 30 mg/mL. Cellular morphology and vacuolization on day 3 (data not shown) appeared similar to the results seen on day 6 of nanoparticle exposure. Interestingly, despite the gradual decline in resistance in ECIS, there was not a drastic increase in cytoplasmic vacuoles or disruption of ppLE cellular morphology from day 3 to day 6. Lens epithelial cells line the inside of the lens capsule. After cataract surgery, these cells can grow and cause a posterior capsule opacification that requires secondary surgery. ppLE cells were tested to determine how the nanoparticles affected the morphology and resistance of lens epithelial cells. The ECIS results showed that at 30 mg/mL, the nanoparticles are toxic after six days. In the lens capsule, the nanoparticle concentration would be 300 mg/mL, ten times higher than the highest tested concentration. Thus, we postulate that the lens epithelial cells would not survive or cause posterior capsule opacification when exposed to a nanoparticle concentration of 300 mg/mL. This is an ideally preferred situation.

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Figure 9. Light microscope images taken at 10X of pLE cells exposed to nanoparticles for 6 days. These in vitro results would need to be confirmed in vivo to determine the actual effect of escaped and dilute nanoparticles from the lens capsule on the retina. Similarly, in vivo studies would determine the toxicity of the nanoparticles on lens epithelial cells and the presence of posterior capsule opacification following lens removal and nanoparticle addition to the lens capsule.

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Conclusions In this study we looked at polyacrylamide copolymers that we crosslinked by forming amides with diamines. The composition of copolymers and the conditions for crosslinking were optimized to form single-molecular nanoparticles that would closely match the properties of βhigh lens crystallins in refractive index, size, intrinsic viscosity, and dynamic viscosity. The refractive indices of the nanoparticle solution at different concentrations matched that of the βhigh lens crystallins. The viscosity of the nanoparticles was lower than un-crosslinked polymers, and approached that of the βhigh lens crystallins. The difference in viscosity between the nanoparticles and the βhigh lens crystallins indicates that the βhigh lens crystallins are still slightly more compact than the nanoparticles. Crosslinking using AA has an advantage over previously studied disulfide-based nanoparticles, because such amide-based crosslinking is not reversible, but is permanent. These materials have potential for use as replacements for the crystallins in developing an accommodating lens hydrogel nanocomposite. The biocompatibility data presented for ppRPE and ppLE cells showed that, in the short term, both cell types tolerated the nanoparticles well at low concentrations. However, at 30 mg/mL, extensive cell death occurred in ppRPE, and there was a marked decrease in ppLE health, as determined by ECIS measurements after six days of exposure. These results were expected, and are acceptable for the type of cell exposure anticipated with these nanoparticles. Further in vivo studies will be needed to validate these in vitro findings. ACKNOWLEDGMENTS 29


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This research was supported by the NIH grant EY021620 and Department of Veterans Affairs Rehab merit review grant RX000657-01 to Dr. Nathan Ravi. Also, Research to Prevent Blindness, Inc. and NIH Core Grant P30 EY02687 facilities supported parts of the research. We thank Mr. Paul Hamilton for conducting the GPC test, and Prof. James Ballard for editing the manuscript. References

1. Glasser, A.; Campbell, M. C. W. Biometric, optical and physical changes in the isolated human crystalline lens with age in relation to presbyopia. Vision Res 1999, 39 (11), 1991-2015. 2. Beers, A. P.; van der Heijde, G. L. Age-related changes in the accommodation mechanism. Optometry and vision science : official publication of the American Academy of Optometry 1996, 73 (4), 235-42. 3. Weale, R. A. On potential causes of presbyopia. Vision Res 1999, 39 (7), 1263-72. 4. Farnsworth, P. N.; Shyne, S. E. Anterior zonular shifts with age. Exp Eye Res 1979, 28 (3), 291-7. 5. Koretz, J. F.; Handelman, G. H. Modeling Age-Related Accommodative Loss in the Human-Eye. Math Modelling 1986, 7 (5-8), 1003-1014. 6. Glasser, A.; Campbell, M. C. Presbyopia and the optical changes in the human crystalline lens with age. Vision Res 1998, 38 (2), 209-29. 7. Koretz, J. F.; Kaufman, P. L.; Neider, M. W.; Goeckner, P. A. Accommodation and presbyopia in the human eye--aging of the anterior segment. Vision Res 1989, 29 (12), 1685-92. 8. Koretz, J. F.; Cook, C. A.; Kaufman, P. L. Accommodation and presbyopia in the human eye. Changes in the anterior segment and crystalline lens with focus. Invest Ophthalmol Vis Sci 1997, 38 (3), 569-78. 9. Reilly, M. A.; Rapp, B.; Hamilton, P. D.; Shen, A. Q.; Ravi, N. Material characterization of porcine lenticular soluble proteins. Biomacromolecules 2008, 9 (6), 1519-1526. 10. Ravi N, Aliyar H, Hamilton PD. Hydrogel Nanocomposite as a synthetic intraocular lens capable of accommodation, Macromol. Symp. 2005, 227 (1), 191-202. 11. Liang, J.; Struckhoff, J. J.; Du, H.; Hamilton, P. D.; Ravi, N. Synthesis and characterization of in situ forming anionic hydrogel as vitreous substitutes. Journal of Biomedical Materials Research Part B: Applied Biomaterials 2016, n/a-n/a. 12. Reilly, M. A.; Hamilton, P. D.; Perry, G.; Ravi, N. Comparison of the behavior of natural and refilled porcine lenses in a robotic lens stretcher. Exp Eye Res 2009, 88 (3), 483-494. 13. Bloemendal, H.; de Jong, W.; Jaenicke, R.; Lubsen, N. H.; Slingsby, C.; Tardieu, A. Ageing and vision: structure, stability and function of lens crystallins. Prog Biophys Mol Bio 2004, 86 (3), 407-485. 30


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14. Lee, C. J.; Vroom, J. A.; Fishman, H. A.; Bent, S. F. Determination of human lens capsule permeability and its feasibility as a replacement for Bruch's membrane. Biomaterials 2006, 27 (8), 1670-1678. 15. Tarcha, P. J. Polymers for Controlled Drug Delivery; CRC Press1990. 16. Higashibayashi, S.; Kohno, M.; Goto, T.; Suziki, K.; Mori, T.; Hashimoto, K.; Nakata, M. Synthetic studies on thiostrepton family of peptide antibiotics: synthesis of the pentapeptide segment containing dihydroxyisoleucine, thiazoline and dehydroamino acid. Tetrahedron Lett 2004, 45 (19), 3707-3712. 17. Tachibana, Y.; Monde, K.; Nishimura, S. I. Sequential glycoproteins: Practical method for the synthesis of antifreeze glycoprotein models containing base labile groups. Macromolecules 2004, 37 (18), 6771-6779. 18. Gartner, Z. J.; Kanan, M. W.; Liu, D. R. Expanding the reaction scope of DNAtemplated synthesis. Angew Chem Int Edit 2002, 41 (10), 1796-+. 19. Chhabra, R.; Sharma, J.; Liu, Y.; Yan, H. Addressable molecular tweezers for DNA-templated coupling reactions. Nano Lett 2006, 6 (5), 978-983. 20. Li, X. Y.; Gartner, Z. J.; Tse, B. N.; Liu, D. R. Translation of DNA into synthetic N-acyloxazolidines. J Am Chem Soc 2004, 126 (16), 5090-5092. 21. Liang, J.; Cheng, L.; Struckhoff, J. J.; Ravi, N. Investigating triazine-based modification of hyaluronan using statistical designs. Carbohyd Polym 2015, 132, 472480. 22. Farkas, P.; Bystricky, S. Efficient activation of carboxyl polysaccharides for the preparation of conjugates. Carbohyd Polym 2007, 68 (1), 187-190.

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Table of Content Graph

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Preparation and Characterization of Biomimetic Î² ... - ACS Publications

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