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polydopamine nanocomplexes to control protein drug release
Accepted Manuscript One-pot synthesis of dopamine-conjugated hyaluronic acid/polydopamine nanocomplexes to control protein drug release Dae Gon Lim, Racelly Ena Prim, Eunah Kang, Seong Hoon Jeong PII: DOI: Reference:
S0378-5173(18)30153-4 https://doi.org/10.1016/j.ijpharm.2018.03.007 IJP 17350
To appear in:
International Journal of Pharmaceutics
Received Date: Revised Date: Accepted Date:
18 October 2017 2 February 2018 6 March 2018
Please cite this article as: D.G. Lim, R.E. Prim, E. Kang, S.H. Jeong, One-pot synthesis of dopamine-conjugated hyaluronic acid/polydopamine nanocomplexes to control protein drug release, International Journal of Pharmaceutics (2018), doi: https://doi.org/10.1016/j.ijpharm.2018.03.007
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One-pot synthesis of dopamine-conjugated hyaluronic acid/polydopamine nanocomplexes to control protein drug release
Dae Gon Lim1, Racelly Ena Prim1, Eunah Kang2*, Seong Hoon Jeong1*
1
College of Pharmacy, Dongguk University-Seoul, Goyang, Gyeonggi, Republic of Korea
2
School of Chemical Engineering and Material Science, Chung-Ang University, Seoul,
Republic of Korea
* To whom correspondence should be addressed. Seong Hoon Jeong, PhD College of Pharmacy Dongguk University – Seoul Goyang, Gyeonggi 13026, Republic of Korea Tel: 82) 10-5679-0621 E-mail: [email protected]
Eunah Kang, PhD School of Chemical Engineering and Material Science Chung-Ang University 221 Heukseok-Dong, Dongjak-Gu, Seoul, Korea Tel: 82)-2-820-6684 E-mail: [email protected]
Submitted to International Journal of Pharmaceutics
Abstract The self-organizing complexes with hyaluronic acid (HA) and polydopamine (PDA), an adhesion mediator via hydrogen bonding, were investigated for use as protein drug carriers. The complexes were prepared with HA of different molecular weights (20 kDa and 200 kDa) and various molar ratios of dopamine and lysozyme, a model protein. Dopamine-conjugated HA (HADA)/PDA complexes were prepared by one-pot synthesis by relying on the selfpolymerization of dopamine under oxidative, weakly basic conditions. Lysozyme was bound via coacervation and hydrogen bonding into HADA/PDA complexes. Complex diameters were 100–300 nm, based on transmission electron microscopy image and dynamic light scattering findings. Circular dichroism and differential scanning calorimetry showed that a stable protein formulation was obtained without degradation while preserving the thermal characteristics of lysozyme. Transition temperature (Tm) of the HADA/PDA/lysozyme complex (1:10:0.05 ratio) was 72.45 oC, which is close to the Tm of the native lysozyme (72.46 oC). The efficacy of complexes was also evaluated to protect the structural stability of lysozyme. Lysozyme (0.33 mol) was complexed with HA monomer; consequently, lysozyme activity in the HADA/PDA complex was not affected from short-term degradation. Protein encapsulation and efficacy of the formulations showed successful complexation as protein carriers, thus suggesting an effective combinatory protein delivery system.
1. Introduction Hyaluronic acid (HA), a linear nonsulfated glycosaminoglycan which is predominant in the extracellular matrix, controls tissue hydration via its high hydrophilicity and high water-holding
capacity
(Luo
and
Prestwich,
2001;
Mero
and
Campisi,
2014;
Papakonstantinou et al., 2012; Solis et al., 2012; Tripodo et al., 2015). It is an ideal biopolymer for bioconjugation due to its excellent biocompatibility, specific targeting, and high drug-loading capacity (Cyphert et al., 2015; Mero and Campisi, 2014), making it safe and good for targeted drug delivery studies (Necas et al., 2008; Water et al., 2014) even with a protective coating (Luo and Prestwich, 2001; Tripodo et al., 2015). Moreover, HA specifically binds to cancer cells that overexpress CD44 (the HA receptor) (Avigdor et al., 2004; Ganesh et al., 2013), and thus, various HA-anti cancer drug conjugates and drugloaded HA nanoparticles (Choi et al., 2011; Thomas et al., 2015) have been investigated for their potential to improve drug targeting and to enhance the therapeutic efficacy of cancer drugs (Choi et al., 2010; Choi et al., 2009; Yoon et al., 2012). Polydopamine (PDA) thin films, formed by self-polymerization of dopamine (DOPA) under a weakly basic condition, have now become one of the commonly used coatings for nanoparticle formation through the mussel adhesion-inspired chemistry (Huang et al., 2015; Hui et al., 2014; Liu et al., 2016). Due to its good stability in the physiological environment, DOPA is also recognized as a promising carrier to improve drug transport and distribution by taking advantages of self-polymerization capabilities and good adhesion properties (Jiang et al., 2011; Kong et al., 2016). Diverse reactive groups of DOPA can be conjugated further with other biomolecules for surface functionalization (Park et al., 2014; Wu et al., 2016). The progress in the mussel-inspired chemistry has extended the biomedical and drug delivery applications of PDA-based nanoparticles as photothermal agents, drug-loading platforms, and
bioimaging and biosensing materials (Dong et al., 2016; Lee et al., 2016a; Liu et al., 2016; Madhurakkat Perikamana et al., 2015). With all the characteristics and benefits of HA and DOPA, the HA complexes combined with PDA may provide specific binding and increase physical stability, wherein DOPA may activate local adhesion effect through HA chains (Luo and Prestwich, 2001; Water et al., 2014; Wu et al., 2016). The new design of HA/DOPA derivatives as nanocomplexes or complexes for protein therapeutics will be useful for modulating their pharmacokinetic and pharmacodynamic profiles (Lee and Cho, 2017; Yang et al., 2012). In this
study,
dopamine-conjugated
hyaluronic
acid/polydopamine
(HADA/PDA)
nanocomplexes were developed by “one-pot synthesis” which includes lysozyme as a model protein drug to bind with HA by complex coacervation (Water et al., 2014). The HADA/PDA complexes loaded with protein can be employed to improve drug delivery efficacy and to preserve protein therapeutic activity. This study aims to develop stable and effective HADA/PDA complexes for controlling protein delivery. The physical properties of the complexes were characterized by dynamic light scattering (DLS), transmission electron microscopy (TEM), circular dichroism (CD), and differential scanning calorimetry (DSC). Furthermore, along with the encapsulation efficiency and protein activity, protein delivery efficacy was also evaluated.
2. Materials and Method 2.1. Reagents and substrate Sodium hyaluronate (MW 20 kDa and 200 kDa) was obtained from Lifecore Biomedical (Chaska, USA). Dopamine HCl, lysozyme from chicken egg white, Nhydroxysuccinimide
(NHS),
1-ethyl-3-[3-dimethylaminopropyl]
carbodiimide
(EDC),
hyaluronidase (HAdase) from bovine testes, Trizma HCl, Trizma base, potassium phosphate
monobasic, and the substrate Micrococcus lysodeikticus were all purchased from SigmaAldrich (St. Louis, USA). Potassium hydroxide was obtained from Daejung Chemicals (Seoul, Korea). All other reagents used were of analytical or HPLC grade.
2.2. Synthesis of NHS-Hyaluronic acid Sodium hyaluronate (100 mg or 250 µmole) was dispersed in 50 mL of distilled water and then adjusted to pH 3.0 using hydrochloric acid (1N and 0.1N). EDC (55 µmole) was dissolved in the sodium hyaluronate solution. After 10 min, NHS (50 µmole) was dissolved in the hyaluronate solution and then reacted for 24 h using a MyLab multimixer SLRM-3 (SeouLin Bioscience, Korea). After the reaction, the NHS-HA solution was dialyzed at 4°C for 12 h using a Biotech cellulose ester membrane (molecular weight cutoff 50 kDa) (Spectrum Laboratories, Inc., USA) in 10 mM phosphate buffer (pH 6.0). The dialyzed HANHS was lyophilized using a Lyopride 20R lyophilizer (Ilshin Biobase, Korea) for further studies (Scheme 1). The functionalization of HA-NHS was identified using a Varian VNS 600 MHz NMR spectrometer (Agilent Technologies, USA). Samples for
1
H-NMR
measurement were dissolved in D2O at 5 mg/mL and then used to fill disposable NMR inserts.
2.3. Preparation of HADA/PDA and HADA/PDA/lysozyme complexes The HADA/PDA complex was formed by adding HA (20 kDa and 200 kDa) with various molar ratios of DOPA (HA:DOPA, 1:1, 1:2, 1:5, and 1:10) in 10 mM Tris buffer (pH 7.5 and 8.5). Samples were placed in a MyLab multimixer SLRM-3 in a cold room for 1 h for optimal DOPA polymerization and HADA/PDA complex formation. HADA/PDA/lysozyme complexes ranged from 0.005 to 0.05 protein ratios were also prepared with the similar method of HADA/PDA complex preparation. The predetermined amount of DOPA was
dissolved in the HA-NHS (20 kDa and 200 kDa) solution (200 μg/mL) in 10 mM Tris buffer (pH 8.5). After 5 min of stirring, the predetermined amount of lysozyme was added to the HADA/PDA solution. The solution was stirred in a cold room (4 oC) for 1 h to prevent protein degradation and to allow for the formation of stable complexes.
2.4. Physical characterization of HADA/PDA and HADA/PDA/lysozyme complexes Particle size and zeta potential were measured using a Zetasizer Nano ZS90 apparatus (Malvern Instruments, UK). Each sample (1 mL) was analyzed in a disposable sizing cuvette (Kartell Labware, Italy) for particle size determination and in a disposable folded capillary cell (Malvern Instruments, UK) for zeta potential. Samples were equilibrated at 25°C and were measured three times at a fixed angle of 90°. The morphology of PDA, HADA/PDA, and HADA/PDA/lysozyme complexes were observed by TEM, using a JEM 2100F field emission transmission electron microscope (Jeol Ltd., Japan), at 200 kV acceleration voltage. Before the measurement, diluted samples were dropped onto a copper grid with 200 mesh size and then dried (Jeol Ltd., Japan).
2.5. Structural characterization of HADA/PDA/lysozyme complexes The secondary structure of lysozyme was determined by CD spectroscopy, a Chirascan instrument (Applied Photophysics, UK), equipped with a TC125 temperature controller (Quantum Northwest, USA) set at 25 ºC. The samples were diluted to desired concentration, loaded into 1 mm quartz precision cells (Hellma Analytics, Germany), and scanned three times from 190 nm to 260 nm. Thermodynamic properties of the complexes were evaluated using a Nano DSC (TA Instruments, USA). The samples and reference were first degassed under vacuum in a degassing station (TA Instruments, USA) and then loaded into respective twin capillary cells.
Tris buffer (pH 8.5) was used as the reference. All samples were scanned from 20 to 100 °C with the scanning rate of 1.0 °C/min. Final thermogram and transition melting temperature (Tm) were obtained by integration and modeling using a NanoAnalyze software v3.7 (TA Instruments, USA). All experiments were carried out in triplicate.
2.6. Complexation degree of HADA/PDA complexes The evaluation of protein complexation was performed using a reversed-phase highperformance liquid chromatography (RP-HPLC) to confirm the binding between HA and lysozyme in the complexes (Water et al., 2014). Complexes prepared with various HA concentrations (0.010.05 mg/mL) and fixed amounts of lysozyme and DOPA were evaluated for the degree of complexation (n = 3). The samples were incubated for 1 h and centrifuged at 13,000 rpm for 25 min. The supernatant was analyzed using an Agilent 1100 HPLC system (Agilent, USA), equipped with a TSK-GEL Protein C4-300 column (4.6 mm × 15 cm, 3.5 µm; TOSOH Bioscience, USA) thermostated at 40°C. Chromatographic separation was achieved by gradient elution of mobile phases A (95% H2O/4.9% acetonitrile [ACN]/0.1% trifluoroacetic acid [TFA]) and B (95% ACN/4.9% H2O/0.1% TFA) at 85:15 to 45:55, at a flow rate of 0.75 mL/min for 30 min. The injection volume was 10 µL, and the diode array detector was set at 280 nm. The lysozyme concentration in the supernatant was calculated based on the peak area at the retention time of 16.1 min (Figure S1). The average number of lysozyme molecules per HA chain was calculated using the following formula:
[LZ]free = lysozyme concentration in the supernatant [LZ]initial and [HA]initial = initial lysozyme and hyaluronic acid concentration, respectively, in the sample
2.7. Lysozyme efficacy test Lysozyme was assayed with the substrate M. lysodeikticus to evaluate the efficacy of the HADA/PDA/lysozyme complexes maintaining the lysozyme activity (Alves et al., 2017; Wang et al., 2016). This test relies on the lytic activity of lysozyme against the cell wall of M. lysodeikticus, leading to changes in the turbidity of the suspension. Briefly, 0.02% (w/v) M. lysodeikticus was prepared in 66 mM potassium phosphate buffer pH 6.26 (absorbance of 0.60.7 at 450 nm); 400 U/mL lysozyme in the cold potassium phosphate buffer was used as the control. The substrate suspension (2.5 mL) was placed in a quartz cell (Mecasys Co., Ltd., Korea), and 100 µL lysozyme and the HADA/PDA complexes were added directly to the substrate suspension. To evaluate the stability of complexes, HAdase (100 µg = 40100 U) was added and allowed to cleave the HA from the complex for 10 min. The change in absorbance at 450 nm (ΔA 450 nm) was monitored at 1 min intervals for 5 min using an Optizen Pop UV/Vis spectrophotometer (Mecasys Co., Ltd., Korea). The blank was composed of 2.5 mL substrate suspension and 100 µL potassium phosphate buffer (pH 6.26). All experiments were performed in triplicate. The active lysozyme units were calculated using the following formula: Units/mL enzyme =
–
0.001 = ΔA 450 nm as per the unit definition of lysozyme activity 0.1 = Volume (mL) of enzyme solution
3. Results and Discussion 3.1. HA-NHS crosslinking and HADA/PDA complex
Amine-reactive HA-NHS was prepared by the EDC/NHS reaction. The characteristic peak of 2.9 ppm assigned to NHS was confirmed by 1H-NMR spectroscopy (Figure S2 and Scheme 1a) (Lee et al., 2016b). The HA-NHS (20 kDa and 200 kDa) was obtained as a white cake after lyophilization. HADA/PDA complexes were formed at 1:1, 1:2, 1:5, and 1:10 molar ratios of HA:DOPA at both pH 7.5 and 8.5. Complex formation of HADA/PDA was confirmed by measuring the size and zeta potential of the complexes. Figure 1 shows the particle size of the HADA/PDA complexes using HA-NHS: (a) 20 kDa HA and (b) 200 kDa HA. The complexes at pH 7.5 were significantly aggregated at 1:5 molar ratio of HA:DOPA (1685.7 ± 214.9 and 1123.1 ± 392.8 nm) for both HA-NHS of 20 kDa and 200 kDa, suggesting that HADA/PDA complexes might not be formed. The HADA/PDA complexes at 1:10 molar ratio showed a decreased diameter of 247.8 ± 54.9 and 315.4 ± 23.0 nm (HA-NHS 20 kDa and 200 kDa, respectively), suggesting that PDA nanoparticle formation contributed to the formation of HADA/PDA complexes. The formation of HADA/PDA complexes at pH 8.5 was more stable than at pH 7.5, showing a consistent diameter range (243.4558.5 nm) without abnormal aggregation for both molecular weights of HA (20 kDa and 200 kDa). Dopamine at pH 8.5 of weak basic condition is known to undergo self-polymerization and form nanoparticles (Hopper et al., 2014; Lee et al., 2007). The formation of stable HADA/PDA complexes at various HA:DOPA ratios at pH 8.5 was induced by the conjugation with HA-NHS and DOPA selfpolymerization (Scheme 1b). At pH 8.5, the complexes with 1:5 and 1:10 molar ratio of HA:DOPA yielded small and stable complexes ranging from 243.4 to 308.9 nm in hydrodynamic diameter for both 20 kDa and 200 kDa HA. Thus, stable HADA/PDA complexes were successfully prepared by incorporating amine-reactive HA-NHS with selfpolymerizing PDA by one-pot synthesis.
Scheme 1. (a) Chemical conjugation of dopamine-conjugated HA (HADA) and (b) schematic view of HADA/PDA/lysozyme complex formation. The complex was formed by one-pot synthesis by relying on the self-polymerization of dopamine under oxidative, weakly basic condition
Figure 1. Particle size of the HADA/PDA complexes in various molar ratios of HA, (a) 20 kDa and (b) 200 kDa, compared to dopamine at different pH (mean ± SD, n = 3).
3.2. HADA/PDA complexes as protein carriers HADA/PDA complexes loaded with lysozyme as a model protein were prepared at various molar ratios and different pH values (7.5 and 8.5). The complexes were formed from the NHS-activated HA with molecular weights of 20 kDa and 200 kDa. Figure 2 showed the changes in hydrodynamic size with increases in the amount of lysozyme and dopamine. The particle size of 20 kDa HADA complexes (Figure 2a and 2b) was smaller than that of 200 kDa HADA complexes (Figure 2c and 2d) when prepared at pH 8.5. In agreement with the HADA (20 kDa)/PDA complexes without loaded protein, HADA (20 kDa)/PDA/lysozyme prepared at pH 8.5 provided smaller size than at pH 7.5. Increasing the lysozyme content of HADA (20 kDa)/PDA/lysozyme at pH 7.5 significantly induced the aggregation of complexes showing diameters up to 576.3 nm at the ratio of 1:10:0.05, respectively. In a one pot synthesis, DOPA self-polymerization and DOPA-HA conjugation occurred, forming protein HADA/PDA complexes. This might suggest that the core formation of the protein nanocomplexes was induced by electrostatic interaction with negatively charged HA and hydrogen bonding with PDA. Positively charged lysozymes have been reported to form complex coacervates via electrostatic interactions with negatively charged HA (Water et al., 2014). The presence of PDA
within complexes provided additionally interactive driving force to form lysozyme/HADA/PDA complexes in one-pot synthesis. The negative zeta potentials of the complexes might indicate colloidal stability. HADA (20 kDa)/PDA/lysozyme prepared at pH 8.5 showed a higher negative zeta potential than that prepared at pH 7.5 (Figure S3). The higher negative surface charge at pH 8.5 provided stable colloidal dispersion and smaller particle size of the complex. HADA (20 kDa)/PDA/lysozyme
with different molar ratios of the complexes at pH 8.5 were used for further evaluation of protein efficacy as they yielded a desirable size range. The pH 8.5 may provide a suitable environment for synthesizing complexes from amine-reactive linear HA and DOPA. Due to the oxidation of DOPA in a weakly basic condition (Liu et al., 2016), DOPA derivatives formed oligoaggregation by π–π stacking (Lynge et al., 2011) and underwent oxidative polymerization by using the oxygen present in the environment (Jiang et al., 2011). The oxygen-induced polymerization of DOPA formed a durable layer through covalent and non-covalent interactions together with the conjugation between DOPA and HA-NHS (Park et al., 2014). The size dependency of HA might have affected the physical properties of the protein nanocomplexes, which mainly affected their activity at a biological level, such as receptor affinity, cell uptake, and intracellular signaling (Cyphert et al., 2015; Papakonstantinou et al., 2012). With the above properties, morphology, protein secondary structure, and thermal characteristics of HADA/PDA complexes were investigated further.
Figure 2. Particle size of the complexes formed using various molar ratios of HA (20 kDa)/dopamine/lysozyme (a and b) and HA (200 kDa)/dopamine/lysozyme (c and d) at different pH (mean ± SD, n = 3).
3.3. Morphology of HADA/PDA complexes The morphologies of PDA, HADA/PDA, and HADA/PDA/lysozyme complexes (Figure 3) were evaluated by TEM. Representative PDA, HADA/PDA (1:10), and HADA/PDA/lysozyme (1:10:0.05) complexes prepared at pH 8.5 were examined. For PDA nanoparticles (Figure 3a), homogeneous rounded ones were observed in the magnified image (inset of Figure 3a), showing the consistent result with the previous studies of PDA
nanoparticles (Yan et al., 2013; Yildirim and Bayindir, 2014). The HADA/PDA complexes appeared as irregular shapes with elongated connecting chains attached to some of the rounded PDA particles (Figure 3b). The same appearance was also observed for the HADA/PDA/lysozyme complexes (Figure 3c). The images confirmed how linear HA-NHS with the DOPA molecule was formed into the shape of complexes in one-pot synthesis.
Figure 3. TEM images of (a) polydopamine (PDA) nanoparticles with an inset image of magnified PDA nanoparticle, (b) hyaluronic acid/polydopamine (HADA/PDA) (1:10), and (c) hyaluronic acid/polydopamine/lysozyme (HADA/PDA/lysozyme) complexes (1:10:0.05).
3.4. Changes in protein secondary structure The changes of lysozyme secondary structure in HADA/PDA/lysozyme complexes were evaluated using CD. The CD spectra of the complexes at various ratios of HADA/PDA/lysozyme were compared to the native α-helix structure of lysozyme (Figure 4). Two minimum peaks of the native lysozyme at 207 nm and 229 nm provided the features of polypeptides with a highly helical structure. The high amplitude at 191 nm indicated stable tertiary structure of lysozyme. At a low concentration of DOPA (HADA/PDA = 1:5) (Figure 4a), the minimum peak of lysozyme was shifted from 215 nm to 212 nm as the complexes
formed with the protein. The results indicated that the conformation of lysozyme on the outer surface of the nanocomplexes was altered. Specifically, HADA/PDA/lysozyme (1:5:0.005) containing the lowest protein concentration showed reduced curvature and a shifted minimal peak to 210 nm compared to the native lysozyme. On the other hand, the higher lysozyme concentration (1:5:0.05) showed a similar shape of CD spectra of lysozyme, which might be resulted from the high lysozyme ratio in the nanocomplex (Greenfield, 2006). At high DOPA concentration (HADA/PDA = 1:10) (Figure 4b), the amplitude of the bands at 215 nm also decreased as the protein concentration increased. At the low DOPA concentration (HADA/PDA = 1:10), the spectral change among the samples provided consistent results as HADA/PDA/lysozyme complexes at the high DOPA concentration. Spectral changes indicated that an attractive interaction had occurred between the protein and the HADA/PDA complexes. The CD spectra provided information on the secondary structure on the outer surface of the complexes (Miles and Wallace, 2016). The structure of lysozyme might be flattened on the outer surface or incorporated into the HADA/PDA complexes. Steady spectral changes at the high DOPA concentration indicated that this concentration supported the stable incorporation of lysozyme into the complexes and the formation of consistent HADA/PDA complexes (Li et al., 2015). The quantitative protein concentration was particularly required for the reliable determination of the protein configuration by CD (Kelly et al., 2005). Like lysozyme, the presence of DOPA also affected the CD spectra, showing different intensity patterns and peak shift at different DOPA concentrations. The result showed that HADA and PDA modified the structural arrangement of lysozyme within the complexes.
Figure 4. CD spectra of HADA/PDA/lysozyme nanocomplexes with various molar ratios of lysozyme. (a) low (1:5) and (b) high (1:10) molar ratio of DOPA in the preparation of HADA/PDA/lysozyme complexes
3.5. Thermal properties of HADA/PDA/lysozyme nanocomplexes The thermal properties of lysozyme were investigated by comparing the DSC thermograms. Figure 5 shows the thermograms of the HADA/PDA/lysozyme, lysozyme, and
PDA. The transition temperature (Tm), indicating the thermal stability of lysozyme, is summarized for each complex in Table 1 (Bye and Falconer, 2013; Gill et al., 2010). The characteristic Tm of PDA and lysozyme were 74.65 and 72.46 oC, respectively, showing the consistent result with the previous study (Wu et al., 2015). Mostly, the thermograms of lysozyme and PDA were overlapped with those of the HADA/PDA/lysozyme complexes because the high molar ratio of PDA dominated the heat specificity of the complexes. Importantly, the Tm of the HADA/PDA/lysozyme complexes shifted as the lysozyme molar ratio was increased from 0.005 to 0.05. For the HADA/PDA/lysozyme complex, the Tm shifted from 61.50 to 71.65 oC (HADA:PDA = 1:5) and from 74.81 to 72.45 oC (HADA:PDA = 1:10). The Tm of 74.81 oC for HADA/PDA/lysozyme complex (1:10:0.005) resulted from the PDA at the low concentration of lysozyme, considering that the Tm of PDA is 74.65 oC. Consistently, with the increases in the amount of lysozyme, the Tm of the nanocomplexes approached the Tm of lysozyme itself (72.46 oC). The result indicated that a stable formulation might be achieved without significant degradation while preserving the thermal characteristics of lysozyme. The slightly reduced Tm values of the nanocomplexes resulted from the low transition temperature values of lysozyme and HA (Water et al., 2014). The slight decrease in Tm values did not negatively affect the stability of proteins, rather the result confirmed the successful binding within the nanocomplexes. Moreover, high DOPA concentration supported the stable incorporation of lysozymes into the nanocomplexes and did not impair the thermal stability of protein within the complexes (Lynch and Dawson, 2008).
Figure 5. DSC thermograms of HADA/PDA/lysozyme nanocomplexes as the lysozyme concentration increased: (a) low dopamine molar ratio (HA:DOPA = 1:5), (b) high dopamine molar ratio (HA:DOPA = 1:10)
Table 1. The transition temperature (Tm), indicating the stability of lysozyme against thermal degradation, is summarized for each nanocomplex (mean ± SD, n=3). Lysozyme Concentration Transition Temperature (Tm) (µg/mL) Lysozyme (1000 µg/mL)
72.46 ± 0.03
Polydopamine (180 µg/mL)
74.65 ± 0.18
HA:DOPA (1:5)
35.8
61.50 ± 0.03
71.5
61.14 ± 0.04
143.1
71.45 ± 0.05
357.68
71.65 ± 0.05
35.8
74.81 ± 0.07
HA:DOPA
71.5
71.25 ± 0.18
(1:10)
143.1
72.08 ± 0.08
357.68
72.45 ± 0.04
3.6. Encapsulation efficiency and efficacy of the nanocomplexes The degree of complex formation between lysozyme and HADA/PDA was analyzed after solution depletion; herein, the HA content was varied from 0.01 to 0.05 mg/mL at both high (195 µg/ml) and low (95 µg/ml) DOPA concentrations while the lysozyme amount remained constant (20 µM, 286 µg/mL). Figure 6a shows the degree of lysozyme complexation with the HADA/PDA complexes. The y and x axes represent the ratio of free lysozyme to the initial lysozyme and the ratio of the initial HA to lysozyme, respectively. In the presence of the low DOPA concentration, free lysozyme linearly decreased (slope: 10.00, r2 = 0.9952) as the ratio of HA to lysozyme [(HA)initial/(LZ)initial] was increased. This result indicated that the addition of linear HA with amine-reactive NHS functional groups induced the complex formation of nanoclusters, which is consistent with the TEM images. At a high DOPA concentration, the ratio of free lysozyme to initial lysozyme became saturated at a low ratio (0.05) of [HA]initial/[LZ]initial indicating that complex formation with HA and DOPA using one-pot synthesis had occurred, leading to efficient encapsulation of lysozyme. On a quantitative mole basis, the binding stoichiometry determined that 16.5 lysozyme molecules could bind per HA chain (20 kDa) or 0.33 moles per HA monomer at saturation. Protein activity on the outer surface of HADA/PDA/lysozyme complexes was investigated by the lysozyme turbidity assay. The enzymatic activity of lysozyme was examined for each formulation. Figure 6b shows the efficacy of the lysozyme complexes as a function of the DOPA concentration and the presence of HAdase. The HADA/PDA/lysozyme
complexes prepared with a low DOPA concentration yielded the highest lysozyme activity when exposed to HAdase. Hence, the degradation of HA by HAdase caused the high exposure of lysozyme to the M. lysodeikticus substrate. In the complexes prepared high DOPA concentrations, there was only 10% increase of active lysozyme at the lysozyme concentration of 0.05 (molar ratio) in the presence of HAdase. Complex coacervates of HA/lysozyme formed by the electrostatic charge balance between the negatively charged HA and the positively charged lysozyme have been reported that protein activity was maintained without significant difference to the native lysozyme (Agarwal et al., 2017). It was assumed that the HADA/PDA complex provided the additional driving force of hydrogen bonding from PDA to load lysozymes within the complexes. The high molar ratio of DOPA mainly induced π-π stacking aggregate domains during the complex formation, consequently suggesting that lysozyme might possibly be loaded within the complexes rather than on the outer surface. The same results were
shown at various lysozyme concentrations. Table 2 summarizes the percentage of lysozyme activity in the complexes (1 mg = 40,000 U), presenting the relative amount of lysozyme on the outer surface of the nanocomplexes compared to the total loaded lysozyme. The low efficacy of lysozyme indicated the successful encapsulation of lysozyme within the HADA/PDA complexes even in the presence of HAdase. Short- and long-term degradation of PDA films has been reported in a multi-enzyme system (Lynge et al., 2011), in the presence of hydrogen peroxide (Liu et al., 2013) and glutathione (Hong et al., 2015), in a PDA-derivate melanin implant system (Bettinger et al., 2009), and in a potential oxidant of NADPH oxidase (Cave et al., 2006). The maintained lysozyme activity in the presence of HAdase provided stable HADA/PDA complexes loaded with protein, which could potentially be degraded in a biocompatible manner. It was reported that HA complexes were polymerized via covalent attachment and formed chemically stable hydrogel complexes (Burdick et al., 2005; Park et al., 2003). However, the HADA/PDA complex was formed via polymerization of dopamine and π–π stacking interaction between HADA and PDA particles. The
HADA/PDA complex was prone to degrade by HAdase and lysozyme activity might be affected due to the change of physical properties (Burdick et al., 2005; Park et al., 2003). Based on the results, a 10 min exposure to HAdase did not completely degrade the complexes as lysozyme activity was not significantly affected by the short-term exposure.
The solution depletion test showed the binding characteristics of HA to lysozyme. The 0.33 moles lysozyme per HA monomer was much higher than the previous studies of approximately 0.1 lysozyme molecule per monomer (Morfin et al., 2011; Water et al., 2014). The effect of DOPA was significant as the complexes with high DOPA concentration reached the saturation level at a low initial HA concentration [(HA)initial]. The limited incorporation of lysozyme depending on the DOPA concentration could be due to the increase of PDA film thickness that was related to DOPA concentration (Ball et al., 2012). The confirmation of protein activity also provided successful complexation of the HADA/PDA complex as protein carriers, demonstrating that HADA/PDA complexes could be used as effective combinatory protein delivery systems.
Figure 6. (a) The degree of complex formation of HADA/PDA/lysozyme by solution depletion study to determine the binding stoichiometry (mean ± SD, n = 3). The HA content was varied from 10 µg/mL to 50 µg/mL at both high (195 µg/ml) and low (95 µg/ml) DOPA concentrations. Lysozyme was used at the constant concentration of 20 µM (286 µg/mL). (b) The comparison of lysozyme units in HADA/PDA/lysozyme nanocomplexes based on high and low dopamine concentrations in the presence of HAdase (mean ± SD, n = 3).
Table 2. The percentage of lysozyme activity in the HADA/PDA/lysozyme complexes (1 mg = 40,000 U, mean ± SD, n = 3). W/O Hyaluronidase W Hyaluronidase Lysozyme concentration Theoretical Experimental % Lysozyme Experimental % Lysozyme (µg/mL) Unit Unit Efficacy Unit Efficacy
HA:DOPA (1:5)
Control
400U
400U
100%
400U
100%
35.8
1431U
670 ± 33
46.82%
530 ± 27
37.04%
71.5
2861U
1000 ± 50
34.95%
960 ± 24
33.55%
143.1
5723U
1530 ± 46
26.73%
1770 ± 89
30.93%
357.68
14307U
2500 ± 76
17.47%
2790 ± 93
19.50%
35.8
1431U
940 ± 59
65.69%
610 ± 18
42.63%
71.5
2861U
940 ± 38
32.86%
1030 ± 26
36.00%
143.1
5723U
1020 ± 51
17.82%
1360 ± 68
23.76%
357.68
14307U
1620 ± 81
11.32%
1800 ± 150
12.58%
HA:DOPA (1:10)
4. Conclusion Improvement of drug delivery systems while maintaining protein stability using nanoparticles might be helpful in optimizing protein therapeutics in the biopharmaceutical field. Among the various implementations for this purpose, the use of nanocomplexes can be an alternative platform due to its simple preparation. This study was to prepare complexes for protein carriers by simple one-pot synthesis. It demonstrated how the ratios and concentrations of HA, DOPA, and the protein affected the conformational and thermal characteristics of the complexes. Moreover, alterations in the conformation of lysozyme due to its incorporation within HADA/PDA complexes suggested the occurrence of minimal bioconjugation and maximal complex formation. HA produced a synergistic effect as it served as a binding factor as well as a backbone for both PDA and the protein. The presence of protein interaction and activity proved the capability of the HADA/PDA complexes as protein carriers. The study provided helpful insights for further protein formulation. Moreover, the combinatory complex of HA and PDA highlighted its versatility as a drug delivery system.
Acknowledgements This work was supported by the National Research Foundation of Korea (NRF) grant, funded by the Korean government (MSIT) (2015R1A1A1A05000942), and the Basic Science
Research Program through the National Research Foundation of Korea (NRF), funded by the Ministry of Science, ICT & Future Planning (NRF-2015R1C1A2A01053307).
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S0378-5173(18)30153-4 https://doi.org/10.1016/j.ijpharm.2018.03.007 IJP 17350
To appear in:
International Journal of Pharmaceutics
Received Date: Revised Date: Accepted Date:
18 October 2017 2 February 2018 6 March 2018
Please cite this article as: D.G. Lim, R.E. Prim, E. Kang, S.H. Jeong, One-pot synthesis of dopamine-conjugated hyaluronic acid/polydopamine nanocomplexes to control protein drug release, International Journal of Pharmaceutics (2018), doi: https://doi.org/10.1016/j.ijpharm.2018.03.007
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One-pot synthesis of dopamine-conjugated hyaluronic acid/polydopamine nanocomplexes to control protein drug release
Dae Gon Lim1, Racelly Ena Prim1, Eunah Kang2*, Seong Hoon Jeong1*
1
College of Pharmacy, Dongguk University-Seoul, Goyang, Gyeonggi, Republic of Korea
2
School of Chemical Engineering and Material Science, Chung-Ang University, Seoul,
Republic of Korea
* To whom correspondence should be addressed. Seong Hoon Jeong, PhD College of Pharmacy Dongguk University – Seoul Goyang, Gyeonggi 13026, Republic of Korea Tel: 82) 10-5679-0621 E-mail: [email protected]
Eunah Kang, PhD School of Chemical Engineering and Material Science Chung-Ang University 221 Heukseok-Dong, Dongjak-Gu, Seoul, Korea Tel: 82)-2-820-6684 E-mail: [email protected]
Submitted to International Journal of Pharmaceutics
Abstract The self-organizing complexes with hyaluronic acid (HA) and polydopamine (PDA), an adhesion mediator via hydrogen bonding, were investigated for use as protein drug carriers. The complexes were prepared with HA of different molecular weights (20 kDa and 200 kDa) and various molar ratios of dopamine and lysozyme, a model protein. Dopamine-conjugated HA (HADA)/PDA complexes were prepared by one-pot synthesis by relying on the selfpolymerization of dopamine under oxidative, weakly basic conditions. Lysozyme was bound via coacervation and hydrogen bonding into HADA/PDA complexes. Complex diameters were 100–300 nm, based on transmission electron microscopy image and dynamic light scattering findings. Circular dichroism and differential scanning calorimetry showed that a stable protein formulation was obtained without degradation while preserving the thermal characteristics of lysozyme. Transition temperature (Tm) of the HADA/PDA/lysozyme complex (1:10:0.05 ratio) was 72.45 oC, which is close to the Tm of the native lysozyme (72.46 oC). The efficacy of complexes was also evaluated to protect the structural stability of lysozyme. Lysozyme (0.33 mol) was complexed with HA monomer; consequently, lysozyme activity in the HADA/PDA complex was not affected from short-term degradation. Protein encapsulation and efficacy of the formulations showed successful complexation as protein carriers, thus suggesting an effective combinatory protein delivery system.
1. Introduction Hyaluronic acid (HA), a linear nonsulfated glycosaminoglycan which is predominant in the extracellular matrix, controls tissue hydration via its high hydrophilicity and high water-holding
capacity
(Luo
and
Prestwich,
2001;
Mero
and
Campisi,
2014;
Papakonstantinou et al., 2012; Solis et al., 2012; Tripodo et al., 2015). It is an ideal biopolymer for bioconjugation due to its excellent biocompatibility, specific targeting, and high drug-loading capacity (Cyphert et al., 2015; Mero and Campisi, 2014), making it safe and good for targeted drug delivery studies (Necas et al., 2008; Water et al., 2014) even with a protective coating (Luo and Prestwich, 2001; Tripodo et al., 2015). Moreover, HA specifically binds to cancer cells that overexpress CD44 (the HA receptor) (Avigdor et al., 2004; Ganesh et al., 2013), and thus, various HA-anti cancer drug conjugates and drugloaded HA nanoparticles (Choi et al., 2011; Thomas et al., 2015) have been investigated for their potential to improve drug targeting and to enhance the therapeutic efficacy of cancer drugs (Choi et al., 2010; Choi et al., 2009; Yoon et al., 2012). Polydopamine (PDA) thin films, formed by self-polymerization of dopamine (DOPA) under a weakly basic condition, have now become one of the commonly used coatings for nanoparticle formation through the mussel adhesion-inspired chemistry (Huang et al., 2015; Hui et al., 2014; Liu et al., 2016). Due to its good stability in the physiological environment, DOPA is also recognized as a promising carrier to improve drug transport and distribution by taking advantages of self-polymerization capabilities and good adhesion properties (Jiang et al., 2011; Kong et al., 2016). Diverse reactive groups of DOPA can be conjugated further with other biomolecules for surface functionalization (Park et al., 2014; Wu et al., 2016). The progress in the mussel-inspired chemistry has extended the biomedical and drug delivery applications of PDA-based nanoparticles as photothermal agents, drug-loading platforms, and
bioimaging and biosensing materials (Dong et al., 2016; Lee et al., 2016a; Liu et al., 2016; Madhurakkat Perikamana et al., 2015). With all the characteristics and benefits of HA and DOPA, the HA complexes combined with PDA may provide specific binding and increase physical stability, wherein DOPA may activate local adhesion effect through HA chains (Luo and Prestwich, 2001; Water et al., 2014; Wu et al., 2016). The new design of HA/DOPA derivatives as nanocomplexes or complexes for protein therapeutics will be useful for modulating their pharmacokinetic and pharmacodynamic profiles (Lee and Cho, 2017; Yang et al., 2012). In this
study,
dopamine-conjugated
hyaluronic
acid/polydopamine
(HADA/PDA)
nanocomplexes were developed by “one-pot synthesis” which includes lysozyme as a model protein drug to bind with HA by complex coacervation (Water et al., 2014). The HADA/PDA complexes loaded with protein can be employed to improve drug delivery efficacy and to preserve protein therapeutic activity. This study aims to develop stable and effective HADA/PDA complexes for controlling protein delivery. The physical properties of the complexes were characterized by dynamic light scattering (DLS), transmission electron microscopy (TEM), circular dichroism (CD), and differential scanning calorimetry (DSC). Furthermore, along with the encapsulation efficiency and protein activity, protein delivery efficacy was also evaluated.
2. Materials and Method 2.1. Reagents and substrate Sodium hyaluronate (MW 20 kDa and 200 kDa) was obtained from Lifecore Biomedical (Chaska, USA). Dopamine HCl, lysozyme from chicken egg white, Nhydroxysuccinimide
(NHS),
1-ethyl-3-[3-dimethylaminopropyl]
carbodiimide
(EDC),
hyaluronidase (HAdase) from bovine testes, Trizma HCl, Trizma base, potassium phosphate
monobasic, and the substrate Micrococcus lysodeikticus were all purchased from SigmaAldrich (St. Louis, USA). Potassium hydroxide was obtained from Daejung Chemicals (Seoul, Korea). All other reagents used were of analytical or HPLC grade.
2.2. Synthesis of NHS-Hyaluronic acid Sodium hyaluronate (100 mg or 250 µmole) was dispersed in 50 mL of distilled water and then adjusted to pH 3.0 using hydrochloric acid (1N and 0.1N). EDC (55 µmole) was dissolved in the sodium hyaluronate solution. After 10 min, NHS (50 µmole) was dissolved in the hyaluronate solution and then reacted for 24 h using a MyLab multimixer SLRM-3 (SeouLin Bioscience, Korea). After the reaction, the NHS-HA solution was dialyzed at 4°C for 12 h using a Biotech cellulose ester membrane (molecular weight cutoff 50 kDa) (Spectrum Laboratories, Inc., USA) in 10 mM phosphate buffer (pH 6.0). The dialyzed HANHS was lyophilized using a Lyopride 20R lyophilizer (Ilshin Biobase, Korea) for further studies (Scheme 1). The functionalization of HA-NHS was identified using a Varian VNS 600 MHz NMR spectrometer (Agilent Technologies, USA). Samples for
1
H-NMR
measurement were dissolved in D2O at 5 mg/mL and then used to fill disposable NMR inserts.
2.3. Preparation of HADA/PDA and HADA/PDA/lysozyme complexes The HADA/PDA complex was formed by adding HA (20 kDa and 200 kDa) with various molar ratios of DOPA (HA:DOPA, 1:1, 1:2, 1:5, and 1:10) in 10 mM Tris buffer (pH 7.5 and 8.5). Samples were placed in a MyLab multimixer SLRM-3 in a cold room for 1 h for optimal DOPA polymerization and HADA/PDA complex formation. HADA/PDA/lysozyme complexes ranged from 0.005 to 0.05 protein ratios were also prepared with the similar method of HADA/PDA complex preparation. The predetermined amount of DOPA was
dissolved in the HA-NHS (20 kDa and 200 kDa) solution (200 μg/mL) in 10 mM Tris buffer (pH 8.5). After 5 min of stirring, the predetermined amount of lysozyme was added to the HADA/PDA solution. The solution was stirred in a cold room (4 oC) for 1 h to prevent protein degradation and to allow for the formation of stable complexes.
2.4. Physical characterization of HADA/PDA and HADA/PDA/lysozyme complexes Particle size and zeta potential were measured using a Zetasizer Nano ZS90 apparatus (Malvern Instruments, UK). Each sample (1 mL) was analyzed in a disposable sizing cuvette (Kartell Labware, Italy) for particle size determination and in a disposable folded capillary cell (Malvern Instruments, UK) for zeta potential. Samples were equilibrated at 25°C and were measured three times at a fixed angle of 90°. The morphology of PDA, HADA/PDA, and HADA/PDA/lysozyme complexes were observed by TEM, using a JEM 2100F field emission transmission electron microscope (Jeol Ltd., Japan), at 200 kV acceleration voltage. Before the measurement, diluted samples were dropped onto a copper grid with 200 mesh size and then dried (Jeol Ltd., Japan).
2.5. Structural characterization of HADA/PDA/lysozyme complexes The secondary structure of lysozyme was determined by CD spectroscopy, a Chirascan instrument (Applied Photophysics, UK), equipped with a TC125 temperature controller (Quantum Northwest, USA) set at 25 ºC. The samples were diluted to desired concentration, loaded into 1 mm quartz precision cells (Hellma Analytics, Germany), and scanned three times from 190 nm to 260 nm. Thermodynamic properties of the complexes were evaluated using a Nano DSC (TA Instruments, USA). The samples and reference were first degassed under vacuum in a degassing station (TA Instruments, USA) and then loaded into respective twin capillary cells.
Tris buffer (pH 8.5) was used as the reference. All samples were scanned from 20 to 100 °C with the scanning rate of 1.0 °C/min. Final thermogram and transition melting temperature (Tm) were obtained by integration and modeling using a NanoAnalyze software v3.7 (TA Instruments, USA). All experiments were carried out in triplicate.
2.6. Complexation degree of HADA/PDA complexes The evaluation of protein complexation was performed using a reversed-phase highperformance liquid chromatography (RP-HPLC) to confirm the binding between HA and lysozyme in the complexes (Water et al., 2014). Complexes prepared with various HA concentrations (0.010.05 mg/mL) and fixed amounts of lysozyme and DOPA were evaluated for the degree of complexation (n = 3). The samples were incubated for 1 h and centrifuged at 13,000 rpm for 25 min. The supernatant was analyzed using an Agilent 1100 HPLC system (Agilent, USA), equipped with a TSK-GEL Protein C4-300 column (4.6 mm × 15 cm, 3.5 µm; TOSOH Bioscience, USA) thermostated at 40°C. Chromatographic separation was achieved by gradient elution of mobile phases A (95% H2O/4.9% acetonitrile [ACN]/0.1% trifluoroacetic acid [TFA]) and B (95% ACN/4.9% H2O/0.1% TFA) at 85:15 to 45:55, at a flow rate of 0.75 mL/min for 30 min. The injection volume was 10 µL, and the diode array detector was set at 280 nm. The lysozyme concentration in the supernatant was calculated based on the peak area at the retention time of 16.1 min (Figure S1). The average number of lysozyme molecules per HA chain was calculated using the following formula:
[LZ]free = lysozyme concentration in the supernatant [LZ]initial and [HA]initial = initial lysozyme and hyaluronic acid concentration, respectively, in the sample
2.7. Lysozyme efficacy test Lysozyme was assayed with the substrate M. lysodeikticus to evaluate the efficacy of the HADA/PDA/lysozyme complexes maintaining the lysozyme activity (Alves et al., 2017; Wang et al., 2016). This test relies on the lytic activity of lysozyme against the cell wall of M. lysodeikticus, leading to changes in the turbidity of the suspension. Briefly, 0.02% (w/v) M. lysodeikticus was prepared in 66 mM potassium phosphate buffer pH 6.26 (absorbance of 0.60.7 at 450 nm); 400 U/mL lysozyme in the cold potassium phosphate buffer was used as the control. The substrate suspension (2.5 mL) was placed in a quartz cell (Mecasys Co., Ltd., Korea), and 100 µL lysozyme and the HADA/PDA complexes were added directly to the substrate suspension. To evaluate the stability of complexes, HAdase (100 µg = 40100 U) was added and allowed to cleave the HA from the complex for 10 min. The change in absorbance at 450 nm (ΔA 450 nm) was monitored at 1 min intervals for 5 min using an Optizen Pop UV/Vis spectrophotometer (Mecasys Co., Ltd., Korea). The blank was composed of 2.5 mL substrate suspension and 100 µL potassium phosphate buffer (pH 6.26). All experiments were performed in triplicate. The active lysozyme units were calculated using the following formula: Units/mL enzyme =
–
0.001 = ΔA 450 nm as per the unit definition of lysozyme activity 0.1 = Volume (mL) of enzyme solution
3. Results and Discussion 3.1. HA-NHS crosslinking and HADA/PDA complex
Amine-reactive HA-NHS was prepared by the EDC/NHS reaction. The characteristic peak of 2.9 ppm assigned to NHS was confirmed by 1H-NMR spectroscopy (Figure S2 and Scheme 1a) (Lee et al., 2016b). The HA-NHS (20 kDa and 200 kDa) was obtained as a white cake after lyophilization. HADA/PDA complexes were formed at 1:1, 1:2, 1:5, and 1:10 molar ratios of HA:DOPA at both pH 7.5 and 8.5. Complex formation of HADA/PDA was confirmed by measuring the size and zeta potential of the complexes. Figure 1 shows the particle size of the HADA/PDA complexes using HA-NHS: (a) 20 kDa HA and (b) 200 kDa HA. The complexes at pH 7.5 were significantly aggregated at 1:5 molar ratio of HA:DOPA (1685.7 ± 214.9 and 1123.1 ± 392.8 nm) for both HA-NHS of 20 kDa and 200 kDa, suggesting that HADA/PDA complexes might not be formed. The HADA/PDA complexes at 1:10 molar ratio showed a decreased diameter of 247.8 ± 54.9 and 315.4 ± 23.0 nm (HA-NHS 20 kDa and 200 kDa, respectively), suggesting that PDA nanoparticle formation contributed to the formation of HADA/PDA complexes. The formation of HADA/PDA complexes at pH 8.5 was more stable than at pH 7.5, showing a consistent diameter range (243.4558.5 nm) without abnormal aggregation for both molecular weights of HA (20 kDa and 200 kDa). Dopamine at pH 8.5 of weak basic condition is known to undergo self-polymerization and form nanoparticles (Hopper et al., 2014; Lee et al., 2007). The formation of stable HADA/PDA complexes at various HA:DOPA ratios at pH 8.5 was induced by the conjugation with HA-NHS and DOPA selfpolymerization (Scheme 1b). At pH 8.5, the complexes with 1:5 and 1:10 molar ratio of HA:DOPA yielded small and stable complexes ranging from 243.4 to 308.9 nm in hydrodynamic diameter for both 20 kDa and 200 kDa HA. Thus, stable HADA/PDA complexes were successfully prepared by incorporating amine-reactive HA-NHS with selfpolymerizing PDA by one-pot synthesis.
Scheme 1. (a) Chemical conjugation of dopamine-conjugated HA (HADA) and (b) schematic view of HADA/PDA/lysozyme complex formation. The complex was formed by one-pot synthesis by relying on the self-polymerization of dopamine under oxidative, weakly basic condition
Figure 1. Particle size of the HADA/PDA complexes in various molar ratios of HA, (a) 20 kDa and (b) 200 kDa, compared to dopamine at different pH (mean ± SD, n = 3).
3.2. HADA/PDA complexes as protein carriers HADA/PDA complexes loaded with lysozyme as a model protein were prepared at various molar ratios and different pH values (7.5 and 8.5). The complexes were formed from the NHS-activated HA with molecular weights of 20 kDa and 200 kDa. Figure 2 showed the changes in hydrodynamic size with increases in the amount of lysozyme and dopamine. The particle size of 20 kDa HADA complexes (Figure 2a and 2b) was smaller than that of 200 kDa HADA complexes (Figure 2c and 2d) when prepared at pH 8.5. In agreement with the HADA (20 kDa)/PDA complexes without loaded protein, HADA (20 kDa)/PDA/lysozyme prepared at pH 8.5 provided smaller size than at pH 7.5. Increasing the lysozyme content of HADA (20 kDa)/PDA/lysozyme at pH 7.5 significantly induced the aggregation of complexes showing diameters up to 576.3 nm at the ratio of 1:10:0.05, respectively. In a one pot synthesis, DOPA self-polymerization and DOPA-HA conjugation occurred, forming protein HADA/PDA complexes. This might suggest that the core formation of the protein nanocomplexes was induced by electrostatic interaction with negatively charged HA and hydrogen bonding with PDA. Positively charged lysozymes have been reported to form complex coacervates via electrostatic interactions with negatively charged HA (Water et al., 2014). The presence of PDA
within complexes provided additionally interactive driving force to form lysozyme/HADA/PDA complexes in one-pot synthesis. The negative zeta potentials of the complexes might indicate colloidal stability. HADA (20 kDa)/PDA/lysozyme prepared at pH 8.5 showed a higher negative zeta potential than that prepared at pH 7.5 (Figure S3). The higher negative surface charge at pH 8.5 provided stable colloidal dispersion and smaller particle size of the complex. HADA (20 kDa)/PDA/lysozyme
with different molar ratios of the complexes at pH 8.5 were used for further evaluation of protein efficacy as they yielded a desirable size range. The pH 8.5 may provide a suitable environment for synthesizing complexes from amine-reactive linear HA and DOPA. Due to the oxidation of DOPA in a weakly basic condition (Liu et al., 2016), DOPA derivatives formed oligoaggregation by π–π stacking (Lynge et al., 2011) and underwent oxidative polymerization by using the oxygen present in the environment (Jiang et al., 2011). The oxygen-induced polymerization of DOPA formed a durable layer through covalent and non-covalent interactions together with the conjugation between DOPA and HA-NHS (Park et al., 2014). The size dependency of HA might have affected the physical properties of the protein nanocomplexes, which mainly affected their activity at a biological level, such as receptor affinity, cell uptake, and intracellular signaling (Cyphert et al., 2015; Papakonstantinou et al., 2012). With the above properties, morphology, protein secondary structure, and thermal characteristics of HADA/PDA complexes were investigated further.
Figure 2. Particle size of the complexes formed using various molar ratios of HA (20 kDa)/dopamine/lysozyme (a and b) and HA (200 kDa)/dopamine/lysozyme (c and d) at different pH (mean ± SD, n = 3).
3.3. Morphology of HADA/PDA complexes The morphologies of PDA, HADA/PDA, and HADA/PDA/lysozyme complexes (Figure 3) were evaluated by TEM. Representative PDA, HADA/PDA (1:10), and HADA/PDA/lysozyme (1:10:0.05) complexes prepared at pH 8.5 were examined. For PDA nanoparticles (Figure 3a), homogeneous rounded ones were observed in the magnified image (inset of Figure 3a), showing the consistent result with the previous studies of PDA
nanoparticles (Yan et al., 2013; Yildirim and Bayindir, 2014). The HADA/PDA complexes appeared as irregular shapes with elongated connecting chains attached to some of the rounded PDA particles (Figure 3b). The same appearance was also observed for the HADA/PDA/lysozyme complexes (Figure 3c). The images confirmed how linear HA-NHS with the DOPA molecule was formed into the shape of complexes in one-pot synthesis.
Figure 3. TEM images of (a) polydopamine (PDA) nanoparticles with an inset image of magnified PDA nanoparticle, (b) hyaluronic acid/polydopamine (HADA/PDA) (1:10), and (c) hyaluronic acid/polydopamine/lysozyme (HADA/PDA/lysozyme) complexes (1:10:0.05).
3.4. Changes in protein secondary structure The changes of lysozyme secondary structure in HADA/PDA/lysozyme complexes were evaluated using CD. The CD spectra of the complexes at various ratios of HADA/PDA/lysozyme were compared to the native α-helix structure of lysozyme (Figure 4). Two minimum peaks of the native lysozyme at 207 nm and 229 nm provided the features of polypeptides with a highly helical structure. The high amplitude at 191 nm indicated stable tertiary structure of lysozyme. At a low concentration of DOPA (HADA/PDA = 1:5) (Figure 4a), the minimum peak of lysozyme was shifted from 215 nm to 212 nm as the complexes
formed with the protein. The results indicated that the conformation of lysozyme on the outer surface of the nanocomplexes was altered. Specifically, HADA/PDA/lysozyme (1:5:0.005) containing the lowest protein concentration showed reduced curvature and a shifted minimal peak to 210 nm compared to the native lysozyme. On the other hand, the higher lysozyme concentration (1:5:0.05) showed a similar shape of CD spectra of lysozyme, which might be resulted from the high lysozyme ratio in the nanocomplex (Greenfield, 2006). At high DOPA concentration (HADA/PDA = 1:10) (Figure 4b), the amplitude of the bands at 215 nm also decreased as the protein concentration increased. At the low DOPA concentration (HADA/PDA = 1:10), the spectral change among the samples provided consistent results as HADA/PDA/lysozyme complexes at the high DOPA concentration. Spectral changes indicated that an attractive interaction had occurred between the protein and the HADA/PDA complexes. The CD spectra provided information on the secondary structure on the outer surface of the complexes (Miles and Wallace, 2016). The structure of lysozyme might be flattened on the outer surface or incorporated into the HADA/PDA complexes. Steady spectral changes at the high DOPA concentration indicated that this concentration supported the stable incorporation of lysozyme into the complexes and the formation of consistent HADA/PDA complexes (Li et al., 2015). The quantitative protein concentration was particularly required for the reliable determination of the protein configuration by CD (Kelly et al., 2005). Like lysozyme, the presence of DOPA also affected the CD spectra, showing different intensity patterns and peak shift at different DOPA concentrations. The result showed that HADA and PDA modified the structural arrangement of lysozyme within the complexes.
Figure 4. CD spectra of HADA/PDA/lysozyme nanocomplexes with various molar ratios of lysozyme. (a) low (1:5) and (b) high (1:10) molar ratio of DOPA in the preparation of HADA/PDA/lysozyme complexes
3.5. Thermal properties of HADA/PDA/lysozyme nanocomplexes The thermal properties of lysozyme were investigated by comparing the DSC thermograms. Figure 5 shows the thermograms of the HADA/PDA/lysozyme, lysozyme, and
PDA. The transition temperature (Tm), indicating the thermal stability of lysozyme, is summarized for each complex in Table 1 (Bye and Falconer, 2013; Gill et al., 2010). The characteristic Tm of PDA and lysozyme were 74.65 and 72.46 oC, respectively, showing the consistent result with the previous study (Wu et al., 2015). Mostly, the thermograms of lysozyme and PDA were overlapped with those of the HADA/PDA/lysozyme complexes because the high molar ratio of PDA dominated the heat specificity of the complexes. Importantly, the Tm of the HADA/PDA/lysozyme complexes shifted as the lysozyme molar ratio was increased from 0.005 to 0.05. For the HADA/PDA/lysozyme complex, the Tm shifted from 61.50 to 71.65 oC (HADA:PDA = 1:5) and from 74.81 to 72.45 oC (HADA:PDA = 1:10). The Tm of 74.81 oC for HADA/PDA/lysozyme complex (1:10:0.005) resulted from the PDA at the low concentration of lysozyme, considering that the Tm of PDA is 74.65 oC. Consistently, with the increases in the amount of lysozyme, the Tm of the nanocomplexes approached the Tm of lysozyme itself (72.46 oC). The result indicated that a stable formulation might be achieved without significant degradation while preserving the thermal characteristics of lysozyme. The slightly reduced Tm values of the nanocomplexes resulted from the low transition temperature values of lysozyme and HA (Water et al., 2014). The slight decrease in Tm values did not negatively affect the stability of proteins, rather the result confirmed the successful binding within the nanocomplexes. Moreover, high DOPA concentration supported the stable incorporation of lysozymes into the nanocomplexes and did not impair the thermal stability of protein within the complexes (Lynch and Dawson, 2008).
Figure 5. DSC thermograms of HADA/PDA/lysozyme nanocomplexes as the lysozyme concentration increased: (a) low dopamine molar ratio (HA:DOPA = 1:5), (b) high dopamine molar ratio (HA:DOPA = 1:10)
Table 1. The transition temperature (Tm), indicating the stability of lysozyme against thermal degradation, is summarized for each nanocomplex (mean ± SD, n=3). Lysozyme Concentration Transition Temperature (Tm) (µg/mL) Lysozyme (1000 µg/mL)
72.46 ± 0.03
Polydopamine (180 µg/mL)
74.65 ± 0.18
HA:DOPA (1:5)
35.8
61.50 ± 0.03
71.5
61.14 ± 0.04
143.1
71.45 ± 0.05
357.68
71.65 ± 0.05
35.8
74.81 ± 0.07
HA:DOPA
71.5
71.25 ± 0.18
(1:10)
143.1
72.08 ± 0.08
357.68
72.45 ± 0.04
3.6. Encapsulation efficiency and efficacy of the nanocomplexes The degree of complex formation between lysozyme and HADA/PDA was analyzed after solution depletion; herein, the HA content was varied from 0.01 to 0.05 mg/mL at both high (195 µg/ml) and low (95 µg/ml) DOPA concentrations while the lysozyme amount remained constant (20 µM, 286 µg/mL). Figure 6a shows the degree of lysozyme complexation with the HADA/PDA complexes. The y and x axes represent the ratio of free lysozyme to the initial lysozyme and the ratio of the initial HA to lysozyme, respectively. In the presence of the low DOPA concentration, free lysozyme linearly decreased (slope: 10.00, r2 = 0.9952) as the ratio of HA to lysozyme [(HA)initial/(LZ)initial] was increased. This result indicated that the addition of linear HA with amine-reactive NHS functional groups induced the complex formation of nanoclusters, which is consistent with the TEM images. At a high DOPA concentration, the ratio of free lysozyme to initial lysozyme became saturated at a low ratio (0.05) of [HA]initial/[LZ]initial indicating that complex formation with HA and DOPA using one-pot synthesis had occurred, leading to efficient encapsulation of lysozyme. On a quantitative mole basis, the binding stoichiometry determined that 16.5 lysozyme molecules could bind per HA chain (20 kDa) or 0.33 moles per HA monomer at saturation. Protein activity on the outer surface of HADA/PDA/lysozyme complexes was investigated by the lysozyme turbidity assay. The enzymatic activity of lysozyme was examined for each formulation. Figure 6b shows the efficacy of the lysozyme complexes as a function of the DOPA concentration and the presence of HAdase. The HADA/PDA/lysozyme
complexes prepared with a low DOPA concentration yielded the highest lysozyme activity when exposed to HAdase. Hence, the degradation of HA by HAdase caused the high exposure of lysozyme to the M. lysodeikticus substrate. In the complexes prepared high DOPA concentrations, there was only 10% increase of active lysozyme at the lysozyme concentration of 0.05 (molar ratio) in the presence of HAdase. Complex coacervates of HA/lysozyme formed by the electrostatic charge balance between the negatively charged HA and the positively charged lysozyme have been reported that protein activity was maintained without significant difference to the native lysozyme (Agarwal et al., 2017). It was assumed that the HADA/PDA complex provided the additional driving force of hydrogen bonding from PDA to load lysozymes within the complexes. The high molar ratio of DOPA mainly induced π-π stacking aggregate domains during the complex formation, consequently suggesting that lysozyme might possibly be loaded within the complexes rather than on the outer surface. The same results were
shown at various lysozyme concentrations. Table 2 summarizes the percentage of lysozyme activity in the complexes (1 mg = 40,000 U), presenting the relative amount of lysozyme on the outer surface of the nanocomplexes compared to the total loaded lysozyme. The low efficacy of lysozyme indicated the successful encapsulation of lysozyme within the HADA/PDA complexes even in the presence of HAdase. Short- and long-term degradation of PDA films has been reported in a multi-enzyme system (Lynge et al., 2011), in the presence of hydrogen peroxide (Liu et al., 2013) and glutathione (Hong et al., 2015), in a PDA-derivate melanin implant system (Bettinger et al., 2009), and in a potential oxidant of NADPH oxidase (Cave et al., 2006). The maintained lysozyme activity in the presence of HAdase provided stable HADA/PDA complexes loaded with protein, which could potentially be degraded in a biocompatible manner. It was reported that HA complexes were polymerized via covalent attachment and formed chemically stable hydrogel complexes (Burdick et al., 2005; Park et al., 2003). However, the HADA/PDA complex was formed via polymerization of dopamine and π–π stacking interaction between HADA and PDA particles. The
HADA/PDA complex was prone to degrade by HAdase and lysozyme activity might be affected due to the change of physical properties (Burdick et al., 2005; Park et al., 2003). Based on the results, a 10 min exposure to HAdase did not completely degrade the complexes as lysozyme activity was not significantly affected by the short-term exposure.
The solution depletion test showed the binding characteristics of HA to lysozyme. The 0.33 moles lysozyme per HA monomer was much higher than the previous studies of approximately 0.1 lysozyme molecule per monomer (Morfin et al., 2011; Water et al., 2014). The effect of DOPA was significant as the complexes with high DOPA concentration reached the saturation level at a low initial HA concentration [(HA)initial]. The limited incorporation of lysozyme depending on the DOPA concentration could be due to the increase of PDA film thickness that was related to DOPA concentration (Ball et al., 2012). The confirmation of protein activity also provided successful complexation of the HADA/PDA complex as protein carriers, demonstrating that HADA/PDA complexes could be used as effective combinatory protein delivery systems.
Figure 6. (a) The degree of complex formation of HADA/PDA/lysozyme by solution depletion study to determine the binding stoichiometry (mean ± SD, n = 3). The HA content was varied from 10 µg/mL to 50 µg/mL at both high (195 µg/ml) and low (95 µg/ml) DOPA concentrations. Lysozyme was used at the constant concentration of 20 µM (286 µg/mL). (b) The comparison of lysozyme units in HADA/PDA/lysozyme nanocomplexes based on high and low dopamine concentrations in the presence of HAdase (mean ± SD, n = 3).
Table 2. The percentage of lysozyme activity in the HADA/PDA/lysozyme complexes (1 mg = 40,000 U, mean ± SD, n = 3). W/O Hyaluronidase W Hyaluronidase Lysozyme concentration Theoretical Experimental % Lysozyme Experimental % Lysozyme (µg/mL) Unit Unit Efficacy Unit Efficacy
HA:DOPA (1:5)
Control
400U
400U
100%
400U
100%
35.8
1431U
670 ± 33
46.82%
530 ± 27
37.04%
71.5
2861U
1000 ± 50
34.95%
960 ± 24
33.55%
143.1
5723U
1530 ± 46
26.73%
1770 ± 89
30.93%
357.68
14307U
2500 ± 76
17.47%
2790 ± 93
19.50%
35.8
1431U
940 ± 59
65.69%
610 ± 18
42.63%
71.5
2861U
940 ± 38
32.86%
1030 ± 26
36.00%
143.1
5723U
1020 ± 51
17.82%
1360 ± 68
23.76%
357.68
14307U
1620 ± 81
11.32%
1800 ± 150
12.58%
HA:DOPA (1:10)
4. Conclusion Improvement of drug delivery systems while maintaining protein stability using nanoparticles might be helpful in optimizing protein therapeutics in the biopharmaceutical field. Among the various implementations for this purpose, the use of nanocomplexes can be an alternative platform due to its simple preparation. This study was to prepare complexes for protein carriers by simple one-pot synthesis. It demonstrated how the ratios and concentrations of HA, DOPA, and the protein affected the conformational and thermal characteristics of the complexes. Moreover, alterations in the conformation of lysozyme due to its incorporation within HADA/PDA complexes suggested the occurrence of minimal bioconjugation and maximal complex formation. HA produced a synergistic effect as it served as a binding factor as well as a backbone for both PDA and the protein. The presence of protein interaction and activity proved the capability of the HADA/PDA complexes as protein carriers. The study provided helpful insights for further protein formulation. Moreover, the combinatory complex of HA and PDA highlighted its versatility as a drug delivery system.
Acknowledgements This work was supported by the National Research Foundation of Korea (NRF) grant, funded by the Korean government (MSIT) (2015R1A1A1A05000942), and the Basic Science
Research Program through the National Research Foundation of Korea (NRF), funded by the Ministry of Science, ICT & Future Planning (NRF-2015R1C1A2A01053307).
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