Enzyme-triggered- and tumor-targeted delivery with tunable, methacrylated poly(ethylene glycols) and hyaluronic acid hybrid nanogels

Figures & data

Figure 1. Overview of strategy in synthesis of hybrid network MEO₂MA-OEGMA-MeHa-based NGs with enzymatically sensitive DEGDA crosslinker.

Table 1. Composition (mM) of enzymatically sensitive hybrid network nanogels.

Sample	MeHA	DEGDA	MEO2MA	OEGMA
MEO2MA-OEGMA 15 % -MeHA-DEGDA	11.00	4.00	40.00	9.50
MEO2MA-OEGMA 30 % -MeHA-DEGDA	11.00	4.00	30.00	19.00
MEO2MA-OEGMA 30 % - DEGDA	0.00	4.00	41.00	18.50
MeHA- DEGDA	11.00	13.00	0.00	0.00

The content of the MeHA/DEGDA network is constant, and the percentage of OEGMA monomer in relation to the total concentration is 15 and 30%.

Figure 2. (a) Comparison of ¹H NMR spectra of MEO₂MA-OEGMA30%-DEGDA-, MEO₂MA-OEGMA30%-MeHa-DEGDA NGs and MeHa polymer (in D₂O). (b) TEM micrograph exhibiting surface activity of carboxylic groups from MeHa chains in MEO₂MA-OEGMA30% -MeHa-DEGDA NGs obtained after application of 1% uranyl acetate (UA) staining protocol.

Table 2. Analysis of tunability of hydrodynamic parameters of hybrid and single network nanogels at dedicated temperatures by varying the content of thermosensitive OEGMA monomer.

Type of nanogel	T / °C	Dh / [nm]	ζ-potential / [mV]	PDI *
MEO2MA-OEGMA(15%)-MeHa-DEGDA	25	91.4	−7.4	0.083
	37	82.0	−8.4	0.111
	45	68.8	−9.9	0.141
MEO2MA-OEGMA(30%)-MeHa-DEGDA	25	121.2	−11.5	0.071
	37	105.7	−13.9	0.046
	45	94.8	−16.4	0.065
MEO2MA-OEGMA(30%)-DEGDA	25	121.2	−10.7	0.011
	37	103.9	−11.2	0.040
	45	99.9	−12.3	0.060
MeHa-DEGDA	25	84.4	−10.5	0.004
	37	83.9	−11,0	0.007
	45	85.5	−10.7	0.054
	Single nanogel		Hybrid nanogel MEO2MA-OEGMA-MeHa-DEGDA
Type of nanogel	MeHa-DEGDA	MEO2MA-OEGMA (30%)-DEGDA	15 % OEGMA (with MeHa)	30% OEGMA (with MeHa
MTX encapsulation efficiency (25 °C, pH 7.4) in (μM·) / in %	100 / 40	85/ 34	70 / 28	110 / 44
DOX encapsulation efficiency (25 °C, pH 7.4) in (μM·) / in %
	84 / 45	93.5 / 36	129 / 69	187 / 74.
Dh [nm] / NGs with DOX, 25 °C	98.5	145.5	109.2	135.7
Dh [nm] / NGs with MTX, 25 °C	117.5	148.6	129.6	141.5

* PDI – Polydispersity Index.

Figure 3. (a) Plots of hydrodynamic diameter, D_h, vs temperature, obtained for MEO₂MA-OEGDA30%-MeHa-DEGDA (blue line), MEO₂MA-OEGDA15%-MeHa-DEGDA (red line) and MEO₂MA-OEGDA30%-DEGDA (dark line). (b) Dependences of D_h on temperature obtained at various pH and referred to an aggregation process. MEO₂MA-OEGDA- MeHa-DEGDA at pH 7.4 (green line), 5.0 (pink line), 9.0 (blue line), and MEO₂MA-OEGDA-DEGDA at pH 5.0 (dark line). (c) Plots of zeta potential vs. temperature for increased carboxylic content in the MeHa-based network. (d) Quasi-reversible cycles of polydispersity index (PDI) changes seen at physiological- and hyperthermia-used temperatures and low acidic environment (pH 5); state of minimal tendency to NGs aggregation. All measurements were repeated three times. For clarity of the figure a single mean standard error was shown.

Figure 4. TEM micrographs of A) MEO₂MA-OEGMA30%-MeHa-DEGDA NGs (pH 7.4), (B) MEO₂MA-OEGMA30%-MeHa-DEGDA NGs taken 1-day after their treatment with 2 mg·mL⁻¹ hyaluronidase enzyme (HAdase, pH 5.0). (C) MEO₂MA-OEGMA30%-MeHa-DEGDA NGs reveal their HAdase-based enzymatic degradation at pH 5.0 after 1-week treatment.

Figure 5. (a) Dependencies of light scattering intensity ratio, I_t/I_o, on degradation time of MEO₂MA-OEGMA30%-MeHa-DEGDA NGs at 37 °C in presence and absence of HAdase. Subscripts “0” and “t” represent time t = 0 and t = t, respectively. (b) Distribution of MEO₂MA-OEGMA30%-MeHa-DEGDA NGs size presented as changes in scattered light intensity after various treatment times by HAdase enzyme. (c) Time dependencies of scattered light intensity (derived count rate, DCR) and hydrodynamic diameter, for MEO₂MA-OEGMA30%-MeHa-DEGDA NGs after treatment with HAdase (2 mg/ml) and at pH 7.4 and 5.0. All measurements were repeated three times. For clarity of the figure a single mean standard error was shown.

Figure 6. Profiles of DOX and MTX release from MEO₂MA-OEGMA30%-MeHa-DEGDA NGs during enzymatic degradation at buffered solutions at 37 °C; HAdase concentration - 2 mg/ml. All measurements were repeated three times. For clarity of the figure a single mean standard error was shown.

Table 3. Release parameters for MEO₂MA-OEGMA30%-MeHa-DEGDA NGs as optimal for prolonged-, enzyme-triggered, and tumor-targeted NGs activity.

Release conditions *		First order	Higuchi	Korsmeyer-Peppas	Baker and Longsdale
		F = 1 − e^−kt	F = kt^0 ^.5	F = kt^n	F = kt
Release of DOX
pH 7.4, HAdase	R^2	0.9731	0.9181	0.9390	0.8803
	k	0.039 ± 0.002	11.85 ± 0.03	8.18 ± 0.04	0.003 ± 0.0001
	n	–	–	0.613 ± 0.03	–
pH 5.0, HAdase	R^2	0.9518	0.9185	0.9390	0.8934
	k	0.027 ± 0.001	9.48 ± 0.05	6.540 ± 0.03	0.002 ± 0.0002
	n	–	–	0.506 ± 0.02	–
pH 7.4, without HAdase	R^2	0.5231	0.9227	0.9464	0.9425
	k	0.014 ± 0.02	6.08 ± 0.08	8.00 ± 0.04	0.0007 ± 0.0001
	n	–	–	0.416 ± 0.01	–
pH 5.0, without HAdase	R^2	0.4242	0.9227	0.9465	0.9365
	k	0.009 ± 0.02	4.25 ± 0.05	5.60 ± 0.03	0.0003 ± 0.00002
	n	–	–	0.416 ± 0.01	–
Release of MTX
pH 7.4, HAdase	R^2	0.9624	0.9432	0.9455	0.9309
	k	0.0034 ± 0.0003	11.19 ± 0.02	9.91 ± 0.02	0.003 ± 0.0002
	n	–	–	0.535 ± 0.04	–
pH 5.0, HAdase	R^2	0.9149	0.9349	0.9546	0.9149
	k	0.0016 ± 0.0001	8.68 ± 0.06	5.75 ± 0.04	0.001 ± 0.0003
	n	–	–	0.618 ± 0.03	–
pH 7.4, without HAdase	R^2	0.4470	0.8906	0.9274	0.9103
	k	0.0093 ± 0.0004	4.69 ± 0.05	6.795 ± 0.03	0.0004 ± 0.00001
	n	–	–	0.392 ± 0.02	–
pH 5.0, without HAdase	R^2	0.9307	0.9219	0.9652	0.9144
	k	0.0055 ± 0.0002	2.94 ± 0.04	5.75 ± 0.04	0.0001 ± 0.0002
	n	–	–	0.697 ± 0.06	–

* In all models, F is the fraction mass of DOX released at time t according to particular model, and k is the kinetic constant.

Figure 7. Results of MTT assay for MCF-10A and HOF healthy cell lines after 72-h treatment with free DOX, DOX-loaded and drug-free NGs. The red line depicts the threshold of 70% of cell viability in accordance with ISO 10993–5 (ISO 10993–5:2009, 2009). One way ANOVA was used to test for statistical significance. Differences from control sample were marked with *, whereas ** marked differences between groups. The difference was considered significant for P values <05.

Figure 8. Results of MTT assay with MCF-7 cancer cell line and with and without HAdase after 72-h treatment with free DOX, DOX-loaded and drug-free NGs. One way ANOVA was used to test for statistical significance. Differences from control sample were marked with *, whereas ** marked differences between groups. The difference was considered significant for P values <05.

Figure 9. Results of MTT assay with A2780 cancer cell line and with and without HAdase after 72-h treatment with free DOX, DOX-loaded and drug-free NGs. One way ANOVA was used to test for statistical significance. Differences from control sample were marked with *, whereas ** marked differences between groups. The difference was considered significant for P values <05.

Figure 10. (a) Confocal images of cancer- (MCF-7) and health- (MCF-10A) cells obtained after 72-h incubation with DOX-loaded MEO₂MA-OEGMA30%-MeHa-DEGDA NGs. Cell nuclei were stained with Hoechst fluorescent dye (blue color). Red color was emitted by DOX absorbed by cells. In case of cancer cells overlapping fluorescence signals of DOX and Hoechst dye were separated. (b) 3 D projections of MCF-7 and MCF-10A cell nuclei recorded after 72-h treatment with DOX-loaded MEO₂MA-OEGMA30%-MeHa-DEGDA NGs. For each cell line separated fluorescent signals and merged signal are presented. Blue color marks cell nuclei stained with Hoechst dye. Red color indicates presence of DOX. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article).

Table 4. IC₅₀ values for MEO₂MA-OEGMA30%-MeHa-DEGDA NGs with or without hyaluronidase enzyme for selected cell lines.

Cell line	IC50 values (μM)
without hyaluronidase
		DOX	NGs + DOX
MCF10-A		0.04	0.51
MCF-7		0.43	0.35
A2780		0.099	0.086
HOF		0.77	2.78
with hyaluronidase
MCF10-A		0.07	0.30
MCF-7		0.41	0.28
A2780		0.14	0.12
HOF		0.61	1.06

Figure 11. Representative flow cytometry traces characterizing A278 and MCF-7 cell lines after 24 h of incubation of NGs loaded with 0.005 µM DOX.

ISO 10993-5:2009. (2009). Biological evaluation of medical devices. Part 5: tests for in vitro cytotoxicity. http://www.iso.org/iso/catalogue_detail.htm?csnumber=36406.

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