Preparation of SAHNs and ISHNs
Table 1. Physicochemical properties of SAHNs. The data are expressed as the mean ± standard deviation (SD, n = 3).
(A) Stoichiometric ratio of trivalent transition metallic ion and hyaluronic acid
0.3% Hyaluronic acid and FeCl3
Volume of 0.05 M
Fe3+ ion (µL)
|
Stoichiometric ratio
(HA disaccharide : Fe3+)
|
Size (nm)
|
% SD
|
PDI
|
pH value
|
400
|
1 : 0.17
|
542
|
-
|
0.416
|
2.58
|
420
|
1 : 0.18
|
424
|
-
|
0.277
|
2.58
|
440
|
1 : 0.19
|
492
|
-
|
0.352
|
2.57
|
460
|
1 : 0.19
|
479
|
-
|
0.306
|
2.58
|
480
|
1 : 0.20
|
428
|
-
|
0.236
|
2.58
|
500
|
1 : 0.21
|
407
|
-
|
0.214
|
2.57
|
520
|
1 : 0.22
|
392
|
-
|
0.217
|
2.57
|
0.1% Hyaluronic acid and FeCl3
Volume of 0.05 M
Fe3+ ion (µL)
|
Stoichiometric ratio
(HA disaccharide : Fe3+)
|
Size (nm)
|
% SD
|
PDI
|
pH value
|
140
|
1 : 0.18
|
142
|
4.74
|
0.365
|
2.93
|
150
|
1 : 0.19
|
130
|
7.58
|
0.208
|
2.92
|
160
|
1 : 0.20
|
97.8
|
4.28
|
0.212
|
2.90
|
180
|
1 : 0.23
|
88.5
|
3.20
|
0.176
|
2.88
|
200
|
1 : 0.25
|
93.9
|
3.01
|
0.143
|
2.86
|
220
|
1 : 0.28
|
101
|
1.98
|
0.128
|
2.84
|
240
|
1 : 0.30
|
110
|
2.58
|
0.090
|
2.82
|
260
|
1 : 0.33
|
120
|
1.87
|
0.157
|
2.80
|
280
|
1 : 0.35
|
122
|
4.37
|
0.144
|
2.78
|
300
|
1 : 0.38
|
137
|
1.86
|
0.127
|
2.77
|
310
|
1 : 0.39
|
177
|
0.50
|
0.252
|
2.77
|
320
|
1 : 0.40
|
233
|
1.52
|
0.262
|
2.76
|
(B) Characterization of SAHNs at different pH values
pH
|
Size (nm)
|
% SD
|
PDI
|
Zeta potential
(mV)
|
Zeta potential SD
(mV)
|
2.81
|
109
|
1.32
|
0.066
|
-30.3
|
5.84
|
3.00
|
112
|
1.51
|
0.094
|
-31.2
|
7.36
|
4.10
|
149
|
1.06
|
0.063
|
-33.9
|
6.35
|
5.10
|
213
|
1.11
|
0.035
|
-35.8
|
5.35
|
6.25
|
247
|
1.37
|
0.114
|
-44.1
|
5.23
|
7.20
|
267
|
3.63
|
0.167
|
-46.6
|
4.62
|
The data are expressed as mean ± standard deviation (SD, n = 5).
(C) Characterization of SAHNs by molecular weight of HA
Volume of 0.05M
Fe3+ ion (µL)
|
800,000 Da
|
950,000 Da
|
1,500,000 Da
|
3,600,000 Da
|
150
|
139 nm
|
130 nm
|
163 nm
|
N.D.
|
200
|
112 nm
|
108 nm
|
140 nm
|
N.D.
|
250
|
109 nm
|
120 nm
|
164 nm
|
95.2 nm
|
300
|
125 nm
|
137 nm
|
151 nm
|
102 nm
|
350
|
233 nm
|
282 nm
|
144 nm
|
117 nm
|
* N.D. (Not detected): The results did not meet the criteria.
The data are expressed as the mean ± standard deviation (SD, n = 3).
(D) Characterization of SAHNs by concentration of the stabilizer L-glutamic acid
Concentration of glutamic acid (M)
|
Initial
|
1 day
|
2 days
|
4 days
|
7 days
|
Size*
|
% SD
|
Size
|
% SD
|
Size
|
% SD
|
Size
|
% SD
|
Size
|
% SD
|
0.00
|
108.5
|
3.45
|
219.8
|
1.08
|
232.0
|
3.13
|
243.8
|
5.47
|
262.3
|
6.56
|
0.01
|
126.3
|
2.74
|
146.0
|
0.84
|
153.5
|
1.64
|
160.8
|
3.09
|
166.8
|
4.89
|
0.02
|
147.7
|
3.28
|
154.0
|
1.15
|
160.0
|
2.22
|
161.3
|
2.20
|
163.0
|
2.87
|
0.03
|
139.3
|
1.84
|
179.4
|
0.97
|
190.7
|
0.64
|
198.0
|
1.96
|
228.8
|
1.98
|
0.04
|
141.3
|
2.24
|
183.3
|
0.10
|
194.3
|
1.66
|
207.3
|
2.09
|
211.8
|
4.28
|
0.05
|
180.0
|
2.67
|
185.3
|
1.01
|
197.0
|
1.44
|
238.3
|
2.19
|
219.7
|
4.44
|
HA is a natural long-chain polymeric compound based on a disaccharide that is a combination of D-glucuronic acid and N-acetyl-D-glucosamine (Kogan et al. 2007). Each disaccharide, as a monomer, contains one carboxylic group that is engaged in a coordinate bond with iron(Ⅲ) ion (ferric chloride, FeCl3), where the stoichiometric ratio indicates the proportion between the disaccharide and iron(III) ions (i.e. reaction ratio) (Mercê et al. 2002, Kim et al. 2017).
To investigate the effects of the variations in proportion on the preparation of SAHNs, ferric chloride, supplied as iron(III) ions, was added to 15 mL HA solution, and then experiments were conducted with constant stirring of 250 rpm at ambient temperature for 5 min. The methods changed only the stoichiometric ratio (HA disaccharide: iron(III) ions [ferric chloride]), the amount of added iron(III) ion to hyaluronic acid was adjusted from 1:0.17 to 1:0.40, and the results are showed in Table 1A.
As shown in Table 1A(up), particle size varied with the specific ratio of HA disaccharide: iron(III) ions [ferric chloride]. First, to investigate effects of iron(III) ion input variations, the amount of 0.05 M iron(III) ions placed into 0.3% HA solution was adjusted to 400–520 µL. Up the addition of ≤ 410 μL, or ≥ 480 μL 0.05 M iron(III) ions to the HA solution, it was found that bead and thread-like precipitates were formed. In terms of efficiency, it was found that the most appropriate amount of 0.05 M iron(III) ions was between 420 μL and 470 μL without any bead and thread-like precipitate, and more than 0.05 M iron(III) ions tended to result in a slightly smaller particle size and a slightly lower polydispersity index(PDI) in aqueous systems.
As shown in Table 1C, to ascertain the size dependency on the molecular weight of HA, the SAHNs were prepared by the methods described below. However, its methods changed only the molecular weight of HA from 800,000 to 3,600,000 Da. As shown in Table 1C, the molecular weight of HA did not affect particle size. In further studies, changes in particle size for HA concentration occurred.
As shown in Table 1A(bottom), when between 160 and 280 μL of 0.05 M iron(III) ions were added into 0.1% HA solution, no significant variations in particle size were observed, but the addition of between 200 and 300 μL of 0.05 M iron(III) ions resulted in a relatively low PDI, indicating that nanoparticles of uniform size were formed. In contrast, the addition of 300 μL or more of 0.05 M iron(III) ions resulted in a drastic increase in particle size and PDI, whereas the addition of 150 μL or less of 0.05 M iron(III) ions resulted in a slightly larger particle size, but a drastic increase in PDI, which indicated that irregularly sized nanoparticles were formed.
As shown in Figure 2C, this study aimed to determine the possible influence of variation in HA %(w/v) concentration, HA concentrations of 0.3%, 0.2%, and 0.1% were tested. To ensure a constant stoichiometric ratio (HA disaccharide: iron(III) ion = 1:0.19), the volume of added iron(III) ions was adjusted to 146.7, 293.3, and 440 μL, respectively.
The lowest HA concentrations led to significantly smaller particle size, as shown in Figure 2C; we assumed that potentially, the lower HA solution concentration led to relatively lower opportunities for the combined interactions between the HA molecules directly engaged in the generation and growth of particles.
Additionally, to ascertain the physicochemical stability dependency on ionic strength, SAHNs were prepared by the methods described below. The methods changed only the ionic strength by the addition of KOH, NaOH, Na2CO3, and K2CO3 at a constant pH value of below 4.0. Through this preliminary studies, in general, the binding ability was reduced at high ionic strength, mainly owing to the electrostatic binding between the functional group and metal ion, as well as the higher counter ion condensation. This study also showed that the higher ionic strength typically yielded smaller particle sizes.
To ascertain the physicochemical stability dependency on temperature, the SAHNs were prepared using the methods described below. The methods changed the manufacturing temperature from 25°C to 45°C.
Through this preliminary studies, the reference for comparative observation of nanoparticles behaviors was also measured without any temperature variation at a certain time, with regard to instability of nanoparticles with time. It was found that the higher temperature led to a slightly larger particle size, and when the temperature was returned to 25℃, the size of nanoparticles at the exact time of temperature return was equivalent to that of nanoparticles kept at the same temperature. This indicated that the size of the formed HA-iron complex nanoparticles with the variation of the temperature was reversible.
Characterization of optimal and ISHNs
The structure and morphology of the ISHNs visualized by using NTA are shown in Figure 1. The ISHNs were spherical with a relatively angular surface and displayed a less smooth spherical shape. The particle size was approximately 80 nm to 180 nm, with a maximum of 183 nm.
It was assumed that the self-agglomerating nanoparticles formed by the covalent bonding were the μ-oxo form (Mercê et al. 2002) between FeCl3 and COOH group of hyaluronan and irinotecan hydrochloride.
As shown in Table 2, the mean diameter of particle size and the PDI of the SAHNs, determined by using DLS, were 89.8 ± 3.31 nm and 0.17 ± 0.03, respectively, which represented a narrow size distribution. The zeta potential reflects the surface charge that is used to confirm nanoparticle stability. Thus, it is also important for the surface characterization of nanoparticles. The zeta potential of the SAHNs was -31.9 ± 0.32 mV and the surface charge was negative. After lyophilization, the physicochemical characteristics (particle size, size distribution, and zeta potential) of the reconstituted SAHNs were similar to those of the SAHNs.
Table 2. Physicochemical properties of optimal SAHNs and ISHNs. The data are expressed as the mean ± SD (n = 3).
|
Formulation
|
Mean diameter (nm)
|
Polydispersity index (PDI)
|
Zeta potential (mV)
|
Encapsulation efficiency (%)A
|
Drug-loading efficiency (%)B
|
Drug content
(mg/mL)C
|
Before
lyophilization
|
SAHNs
|
89.3 ± 3.31
|
0.17 ± 0.03
|
-31.9 ± 0.32
|
-
|
-
|
-
|
ISHNs
|
93.8 ± 4.48
|
0.19 ± 0.02
|
-36.3 ± 0.28
|
58.3 ± 0.15
|
25.1 ± 0.09
|
1.75 ± 0.11
|
After lyophilization
|
SAHNs
|
87.5 ± 4.09
|
0.20 ± 0.05
|
-31.2 ± 0.25
|
-
|
-
|
-
|
ISHNs
|
95.2 ± 5.56
|
0.21 ± 0.03
|
-36.6 ± 0.41
|
57.3 ± 0.08
|
24.7 ± 0.07
|
1.72 ± 0.29
|
A Encapsulation efficiency (%) = (amount of irinotecan hydrochloride encapsulated in ISHNs / amount of the feed source of irinotecan hydrochloride) × 100%
B Drug loading efficiency (%) = (amount of irinotecan hydrochloride in ISHNs / amount of the feed source and irinotecan hydrochloride) × 100%
C Drug content (mg/mL) = (amount of irinotecan hydrochloride in ISHNs / amount of the feeding material and irinotecan hydrochloride in the 15-mL vial)
Abbreviations: SAHNs, self-agglomerating hyaluronan nanoparticles; ISHN, irinotecan-loaded self-agglomerating hyaluronan nanoparticles.
The mean diameter of the particle size and the PDI of the ISHNs, determined by using DLS, were 93.8 ± 4.48 nm and 0.19 ± 0.02, respectively, which represented a narrow size distribution. The zeta potential of the ISHNs was -36.8 ± 0.28 mV and have a negative surface charge; that is, there was a negative mean charge on the hyaluronan present on the outer surface of the SAHNs and ISHNs. The mean particle size of the ISHNs was much larger than that of the SAHNs (P < 0.05) owing to the presence of irinotecan. The ISHNs were 5–7 nm larger than the SAHNs.
After lyophilization, the physicochemical characteristics (particle size, size distribution, and zeta potential) of the reconstituted ISHNs were similar to those of the ISHNs of before lyophilization, as shown in Table 2.
It is known that particle size alters the pharmacokinetics through the alteration of tissue distribution and excretion (Maeda et al. 2013). Nanoparticles below <200 nm show increased drug accumulation in tumor cells because of the EPR effect (Fang et al. 2011). In our study, given that the size of the SAHNs and ISHNs was maintained at not more than 110 nm, irinotecan accumulation of tumor cells was anticipated to be relatively high. Both the SAHNs and ISHNs were a rigid angular spherical shape, and as they are nanoparticles of HA, had a negative charge (Table 1A). These data suggested that the mixture core of the ISHNs contained an irinotecan with a hydroxyl group and that the outside was made with HA in the μ-oxo form covalently bound to FeCl3(ferric chloride) in the aqueous phase. That is, these nanoparticles such as ISHNs played an important role in the targeting of NSCLC cells. Overall, the zeta potential, particle characteristics, and morphology of the SAHNs and ISHNs were very similar, but the slight difference in particle size was presumably due to the inclusion of irinotecan HCl.
As shown in Table 2, the LE and EE of the ISHNs (n=3) were 25.1% ± 0.09% and 58.3% ± 0.15%, respectively. After reconstitution, the EE and LE of the ISHNs (n=3) were 57.3% ± 0.08% and 24.7% ± 0.07%, respectively, and these results were satisfactory reproducible. The yield of drug fixation reached 50% to 60% in ISHNs. The EE was >58.3% and was independent of the irinotecan content (1.75 mg/mL) for all batches of the ISHNs tested. These data suggested that irinotecan was most conjugated in the HA-agglomerated mixture of the SAHNs. During preliminary studies, we determined that the drug content in the ISHNs was >2 mg/mL; however, to maintain stability over time, we used <1.7 mg/mL of irinotecan for these studies.
Stability studies
To investigate effects of the variation in proportions on the preparation of the HA-iron complex nanoparticles, iron(III) ions were added to 15 mL HA solution, and the experiments were conducted with constant stirring rate at ambient temperature and 250 rpm for 5 min. The methods changed only the stoichiometric ratio (HA disaccharide: iron(Ⅲ) ion [ferric chloride]); the amount of added iron(III) ion to hyaluronic acid was adjusted from 1:0.18 to 1:0.40.
The SAHNs and ISHNs were reconstituted with injection water to obtain solutions at a concentration of 1.7 mg/mL irinotecan hydrochloride.
The results of the stability dependency of ISHNs on the ratio of metal ion was evaluated by their change of size are shown in Figure 2A. The reconstituted ISHNs without stabilizer were physically unstable when stored at 25°C for 14 days. The size of the tested parameters of the nanoparticles (appearance, particle size, and SD) increased when stored at 25°C for 14 days. Overall, they tended to show an increase in size. However, when between 1 : 0.25 to 0.35 of the stoichiometric ratio(HA:FeCl3) were added into 0.1% HA solution, no significant variations in particle size were observed.
The results of the stability dependency of ISHNs on the pH were evaluated by their change in size over 14 days are shown in Figure 2B. Although the particle size was initially determined by pH, there was no change in particle size over time. The reconstituted ISHNs were physically stable in solutions of various pH stored at 25°C for 14 days. As shown in Figure 2B, the nanoparticles showed no increases in the tested parameters (pH, appearance, particle size, and SD) when stored at 25°C for 14 days. Overall, there was no tendency for the parameters to increase.
The results of the stability dependency of ISHNs on the concentration of HA exhibited between 0.1% to 0.3% are shown in Figure 2C were evaluated by their change in size over 14 days. The initial particle size differed depending on the concentration of HA, and was stable at 0.1% HA, but gradually increased over time at concentrations of 0.2% or more. In our previous experiments, we studied the stability dependency on ionic strength and temperature. Although the particle size was initially determined by the ionic strength and temperature, it did not change over time. The reconstituted ISHNs were physically stable in solutions of various ionic strengths when stored at 25°C for 14 days.
The results of the stability analysis for the total height of samples filled in a cylindrical glass cell using Turbiscan Lab® are shown in Figure 3 and represent the solution stability. The results were measured and illustrated as transmission flux (%) because both samples were of low concentration. Accordingly, the results of analysis were presented in the comparison of transmission flux (%) vs. sample height. As shown in Figure 3A, the reconstituted ISHNs without stabilizer were physically unstable in the test of solution when stored at 25°C for 24 h. However, as shown in Figure 3B, the reconstituted ISHNs with glutamic acid as a stabilizer were physically stable in the test of solution when stored at 25°C for 24 h. Through studies of various materials, L-glutamic acid (GA) was adopted as stabilizer; the coordination with unreacted carboxylic groups occurs in the HA chain and prevents any further growth of particles. Thus, the addition of only 0.02 M GA to the ISHNs achieved the optimal results and there was no significant variation in the size parameters at 25℃ for 7 days. That is, the adoption of glutamic acid contributed to marked stabilization, but the concentration of glutamic acid did not influence the stability, except for 0.02 M GA (Table 1D). The data in the table show that higher concentrations of added GA led to a large particle size in the initial step. In particular, the application of 0.02 M GA, as shown in Figure 3B, resulted in smaller and more stabilized uniform size of particles.
In vitro drug release studies
The 21 h in vitro drug release patterns of ISHN 1 (manufactured with 0.02 M ferric chloride), ISHN 2 (manufactured with 0.05 M ferric chloride), and Camptosar® (irinotecan hydrochloride) in pH 7.0 PBS, as measured using the above mentioned dialysis method, are shown in Figure 4 (Zhao et al. 2012, Yang et al. 2013, Cho et al. 2014). The Camptosar® injection released >90% of irinotecan within 2 h, whereas the ISHNs released <10% after 2 h and approximately 75% after 10 h. The ISHNs made with 0.02 M and 0.05 M FeCl3 resulted in a sustained released pattern in which the irinotecan hydrochloride was continuously released from the hyaluronan self-agglomerated nanocarriers, and displayed significantly different release characteristics than those of the Camptosar® injection. Further, the release pattern of the ISHNs made with 0.05 M FeCl3 was sustained for a longer period than that of the ISHNs made with 0.02 M FeCl3. These indicated that the in vitro release of irinotecan hydrochloride was affected by the bonding among hyaluronan, irinotecan, and ferric chloride, determined by the concentration of ferric chloride. We assumed that the ISHNs may circulate and that retention would be higher than for an irinotecan injection into cancer cells for an extended period after administration. Considering the time required for the formulations to reach the cancer cells after administration in the body, a sustained release of the drug would be more advantageous than a rapid-release formulation and offer greater therapeutic benefits (Cho et al. 2014). Therefore, ISHNs may be more efficient for the delivery of drugs via targeted release than Camptosar® injection.
In vitro NSCLC cell-targeting studies
The results of the in vitro NSCLC cell targeting for CD44 exhibited by the SAHNs, which was used to evaluate their capability to target cancer cells that generally overexpress CD44(Taurin et al. 2012) are shown in Figure 5. We used two human NSCLC cell lines, H23 (CD44+) cells, which express CD44 on their surface, and A549 (CD44-) cells, which do not express CD44 (Leung et al. 2010).
The results of the in vitro NSCLC cell uptake of the SAHNs for CD44 receptor, which was performed to assess the cell-targeting capability of these nanoparticles toward human NSCLC cells that generally overexpress CD44, are shown in Figure 5.
The all samples used for the in vitro cell studies were not experimented ISHNs with an irinotecan, because our team could not evaluate the precise cell count as cell death has been observed following the administration of ISHNs containing anticancer agents such as irinotecan hydrochloride in cell-targeting studies.
As a shown in Figure 5(A), the results of the in vitro NSCLC cell affinity studies of the SAHNs for CD44 using A549 (CD44-) cells revealed that specific affinity binding was not observed in all the specimens experimented. The in vitro NSCLC cell affinity test of the SAHNs in A549 (CD44-) cells showed that the SAHNs had no specific binding to CD44 present on the NSCLC cells. In contrast, the SAHNs showed substantial specific uptake binding to H23 (CD44+) cells (Figure 5(A), Bottom). SAHNs had a targeting capability to H23 cells 5-fold higher than that of A549 cells that do not have CD44. Therefore, SAHNs containing HA on the outer-surface of the nanoparticles might offer an outstanding drug delivery system for passive and active tumor targeting.
As a shown in Figure 5(B), the images of the in vitro NSCLC cellular uptake and specific affinity of the SAHNs in the H23 and A549 cells captured by using confocal microscopy. The pictures are of single sections through the A549 and H23 cells. The left picture panels, which show nuclear cell staining, were obtained from the blue cell channel; the center picture panels, which show the fluorescence of encapsulated FITC were obtained from the green channel; and the right picture panels are merged pictures from the previous two images. Although the images of the SAHN-treated A549 cells did not reveal any specific binding and cellular uptake, the SAHNs-treated H23 cells were displayed outstanding cellular uptake and specific affinity binding. The results has demonstrated the theory behind the ligand-receptor reaction between HA and CD44. As shown in Figure 5, the results from SAHNs were consistent with confocal microscopy and FACS data, which suggested that SAHNs with HA facilitates attachment to CD44-overexpressing cells in human NSCLC cells. The results of the FACS and confocal microscopy support that the cell affinity of SAHNs should be sufficient to result in efficient cell binding and uptake via CD44 in NSCLC cells. Therefore, it was shown that the SAHNs with HA are a remarkable drug delivery system for passive and active tumor targeting.