Hersengerichte polysorbaat 80-geëmulgeerde Donepezil geneesmiddelgeladen nanodeeltjes voor neuroprotectie

Resultaten

Kenmerken van nanodeeltjes

CHP aggregeert zichzelf om nanodeeltjes te vormen met hydrofobe kernen, die kunnen worden geladen met DZP. DZP-geladen CHP-nanodeeltjes (DCN's) met geneesmiddel-tot-nanomateriaalverhoudingen van 1:2, 1:5 en 1:10 werden DCN1, DCN2 en DCN3 genoemd. Volgens de resultaten van de scanning-elektronenmicroscopie vertoonden CHP-nanodeeltjes een bolvormige structuur en na DZP-lading waren de DCN's ook bolvormig, zoals weergegeven in Fig. 1. De gemiddelde grootte en zeta-potentiaal van de CHP-nanodeeltjes waren 257 ± 3,05 nm en −2,81 ±  0,27 mV, respectievelijk. Na het laden van DZP waren de gemiddelde grootten 273 ± 3,72, 260,7 ± 1,76 en 266,8 ± 4,56 nm, en de zeta-potentialen waren respectievelijk -6,20 ± 0,40, −5,75 ± 0,64 en -9,30 ± 0,39 mV voor respectievelijk DCN1, DCN2 en DCN3 , zoals weergegeven in tabel 1. De percentages van geneesmiddelinsluiting waren 42,00 ± 5,65%, 86,54 ± 1,31% en 59,71 ± 4,43%, en de percentages geneesmiddelbelading waren 12,02 ± 1,90%, 13,42 ± 2,03% en 7,40 ± 1,72%, respectievelijk.

Scanning elektronenmicroscopie beelden (a , a-CHP, b-DCN1, c-DCN2, d-DCN3), grootteverdeling (b a-CHP, b-DCN1, c-DCN2, d-DCN3) en zeta-potentiaal (c a-CHP, b-DCN1, c-DCN2, d-DCN3) van nanodeeltjes

ITC-meting

Voor DCN's vertoonde de reactie tijdens het gehele reactieproces voornamelijk een opwaartse piek (Fig. 2), en de reactie was endotherm omdat de opwaartse piek een warmteafgevende reactie aangeeft. Daarom kan PS spontaan worden geadsorbeerd op het DCN-oppervlak. De PS-affiniteit was (14,7 ± 2,76) × 10 ⁴ M ⁻¹ , (29.8 ± 1.66) × 10 ⁴ M ⁻¹ en (36,7 ± 3,84) × 10 ⁴ M ⁻¹ , en de mate van PS-dekking was 2,65 ± 0,193, 2,70 ± 0,372 en 1,49 ± 0,434 voor respectievelijk DCN1, DCN2 en DCN3. Dit resultaat geeft aan dat PS met een hoge affiniteit aan het DCN-oppervlak adsorbeerde en een grotere dekking op DCN2 had. ∆H> 0 en ∆S> 0 geeft aan dat de drie deeltjes voornamelijk gebonden waren door hydrofobe interacties met PS.

Isotherme calorimetriegegevens voor PS (0,9 mM) titratie in a DCN1, b DCN2 en c DCN3 (0,02 mM) oplossingen bij 25 °C. Dekkingsgraad, affiniteit (KA) en enthalpie- en entropieveranderingen voor PS-binding met nanodeeltjes (NP's) na titratie in NP-oplossingen

Karakterisering van de drie typen nanodeeltjes

De CHP NP's en DCN's bereid door dialyse vertoonden een uniforme bolvorm (figuur 3a). Volgens de bovenstaande studie kozen we de DZP-CHP-nanodeeltjes met een verhouding tussen geneesmiddelen en nanomaterialen van 1:5 als de onderwerpen van het volgende experiment. De DZP-CHP-nanodeeltjes hadden een relatief uniforme deeltjesgrootte van 260,7 ±-1,76 nm en de dispersie-index was 0,196  ± 0,019. Hoewel de deeltjesgrootte relatief stabiel bleef na het laden van het geneesmiddel, nam deze sterk toe tot 335,2 ±   5,46 nm na PS-adsorptie. De zeta-potentiaal van de DZP-CHP-nanodeeltjes was − 0,66 ± 0,04 mV, en na coating met polysorbaat 80 daalde de zeta-potentiaal tot − 2,22 ± 0,86 mV (Fig. 3b).

Karakterisering van verschillende nanodeeltjes. een a Transmissie-elektronenmicroscopiefoto van WKK NP's, b transmissie-elektronenmicroscopie foto van DZP-CHP NP's, c transmissie-elektronenmicroscopie foto van PS-DZP-CHP NP's. B:Deeltjesgroottediagram en zeta-potentiaaldiagram van WKK-DZP NP's (voedingsverhouding 1:5) en DZP-CHP NP's gemodificeerd met polysorbaat 80 (voedingsverhouding 1:5)

In vitro geneesmiddelafgifte van nanodeeltjes

De resultaten toonden aan dat DZP-CHP NP's en PS-DZP-CHP NP's, vergeleken met gratis donepezil, 72 uur lang DZP afgaven met duidelijke gecontroleerde afgifte-effecten. De vroege snelle afgifte van met geneesmiddel beladen nanodeeltjes kan te wijten zijn aan de snelle oplossing en afgifte van geneesmiddelmoleculen, en dan kan de vertraging worden veroorzaakt door de afname van de geneesmiddelconcentratie, die alleen kan worden beïnvloed door oplossing en diffusie. De in vitro geneesmiddelafgifte van DZP-CHP NP's gecoat en ongecoat met polysorbaat 80 werd vervolgens bestudeerd. De reden voor de langzamere afgifte van PS-DZP-CHP NP's kan te wijten zijn aan de sterke adsorptie van polysorbaat 80 aan kleine hydrofobe moleculaire geneesmiddelen (Fig. 4).

In vitro geneesmiddelafgiftecurves voor DZP, DZP-CHP NP's (1:5) en PS-DZP-CHP NP's

Nanodeeltjes Brain Targeting Effect

Observatie van hersentargeting met behulp van Live Fluorescence Imaging-technologie

De hersenen van muizen die waren geïnjecteerd met gratis ICG vertoonden geen fluorescentie, maar de hersenen die waren geïnjecteerd met nanodeeltjes geëmulgeerd met PS vertoonden een sterkere fluorescentie in de hersenen dan die geïnjecteerd met niet-geëmulgeerde nanodeeltjes omdat beide nanodeeltjes de hersenen konden bereiken na injectie via de staartader (Fig. . 5a). Om dit te verifiëren, hebben we de muizen 30 minuten na intraveneuze injectie van ICG-DZP-CHP- en PS-ICG-DZP-CHP-oplossingen ontleed, alle organen verwijderd die nodig zijn voor het onderzoek en vervolgens fluorescentiebeeldvorming uitgevoerd. PS-ICG-DZP-CHP-nanodeeltjes vertoonden sterke fluorescentie in de hersenen, maar er werd geen waargenomen in andere organen (figuur 5b). Afbeeldingen toonden aan dat nanodeeltjes gemodificeerd met PS de sterkste fluorescentie in hersenweefsel vertoonden, terwijl die niet gemodificeerde zwakke fluorescentie vertoonden. Er werd geen fluorescentie waargenomen in het hersenweefsel van muizen die waren geïnjecteerd met gratis ICG (Fig. 5c).

In vivo fluorescentiebeelden nadat verschillende oplossingen via de staartader waren geïnjecteerd. een Fluorescentiebeelden van hele dieren geïnjecteerd via de staartader met 200 g / ml DZP-CHP of PS-DZP-CHP nanodeeltjes geladen met ICG als een vlek. een gratis ICG-oplossing (fluorescentie-intensiteit × 10 ⁹ ), b ICG-DZP-CHP nanodeeltjes (fluorescentie-intensiteit × 10 ⁷ ), c ICG-DZP-CHP nanodeeltjes gemodificeerd door PS (fluorescentie-intensiteit × 109). b Fluorescence images of various organs after injection of DZP-CHP nanoparticles modified by PS via the tail vein. c Fluorescence images of the brain after dissection. d Brain of mice injected with ICG-PS-DZP-CHP nanoparticles, e brain of mice injected with ICG-DZP-CHP nanoparticles modified with PS, f brain of mice injected with free ICG

Tissue Distribution of Nanoparticles in Mice

Donepezil was distributed in various tissues and mainly in the brain after injection of PS-DZP-CHP nanoparticles. Because it is metabolized through the kidney, the concentration of donepezil is very high in the kidney at certain times (Fig. 6a). In the brain, the concentration of free donepezil reached a peak in a very short time and then decreased rapidly. However, the concentration of donepezil nanoparticles reached a peak much more slowly and then decreased, especially nanoparticles modified with PS, which indicates a sustained-release effect with a delayed peak and prolonged retention time. Obviously, the nanoparticles improved the bioavailability of the drug (Fig. 6b).

een The concentration of donepezil in the brain, heart, liver and kidney at different times. b The concentrations of free DZP, DZP-CHP nanosolution and DZP-CHP nanosolution modified with PS in brain tissue at different times

MST Results

MST results showed that the thermal surge changed regularly with increasing ligand concentration, and the K_D value was 3.63 μM, indicating that the ligand effectively binds to the target protein, which verifies that PS can bind to Apo E and is relatively stable. After surface modification with PS, CHP nanoparticles can promote adsorption of Apo E, theoretically confirming that the nanoparticles we designed can specifically target brain tissue because the nanoparticles adsorbed Apo E, which may mediate passage through the blood–brain barrier (Fig. 7).

een Raw MST data. The fluorescently labeled molecules were observed for 5 s. At this time, the infrared laser was turned on, and a small part of the capillary tube was heated to 2–5 °C. The molecules migrated along a thermal gradient, resulting in changes in fluorescence intensity. When the infrared laser is turned off, the molecules diffuse along the concentration gradient. b The binding curve is generated by the difference between the initial fluorescence intensity and the intensity in the presence of heat, and the curve conforms to the standard 1:1 binding model

Establishment of a Nerve Injury Model Induced by Aβ_25–35

MTT tests were used to detect the effect of different concentrations of Aβ25-35 on the activity of PC12 and SH-SY5Y cells [Fig. 8a (i), Fig. 8b (i)], and the results showed that with increasing Aβ_25–35 concentration, PC12 and SH-SY5Y cell proliferation activity gradually decreased compared with that in the normal control group. When PC12 and SH-SY5Y cells were treated with 20 μM Aβ_25–35 , PC12 cell activity decreased to 49.5 ± 3.3% that observed in the control group (P  < 0.01), and SH-SY5Y cell activity decreased to 49.7 ± 0.8% (P  < 0.01). An LDH kit was used to detect the effect of different concentrations of Aβ_25–35 on LDH activity in both cell supernatants. Colorimetric tests showed that activity in both supernatants increased gradually with increasing Aβ25–35 concentration. After treatment of the cells with 20 μM Aβ_25–35 , PC12 cell LDH release increased to 359.3 ± 18.3% that in the control group (P  < 0.01), and SH-SY5Y cell LDH release increased to 360.0 ± 18.2% (P  < 0.01). Rhodamine 123 staining was used to detect the effect of different concentrations of Aβ_{25– 35} on the mitochondrial membrane potential in both cell lines [Fig. 8a (ii), b (ii)]. The test showed that the mitochondrial membrane potential in both cell lines decreased gradually with increasing Aβ_{25– 35} concentration (5, 10, 20, 40 μmol/L). Treatment of the cells with 20 μM Aβ_25–35 decreased the PC12 cell mitochondrial membrane potential to 51.3 ± 1.6% that in the control group (P  < 0.01); for SH-SY5Y cells, the MMP decreased to 47.9 ± 1.7% that in the control group (P  < 0.01).

Effects of different concentrations of Aβ_25–35 on injury to PC12 and SH-SY5Y cells. een , b The effect of different concentrations of Aβ_25–35 on the cell survival rate, LDH activity and cell mitochondrial membrane potential in PC12 cells and SH-SY5Y cells (*P  < 0.05, ** P < 0.01 vs control group). c The effect of different concentrations of Aβ_{25– 35} on damage to the morphology of PC12 cells:a control group, b Aβ_{25– 35} (5 μM) injury group, c Aβ_{25– 35} (10 μM) injury group, d Aβ_{25– 35} (20 μM) injury group, e Aβ_{25– 35} (40 μM) injury group. D:the effect of different concentrations of Aβ_{25– 35} on injury to the morphology of SH-SY5Y cells:f—control group, g Aβ_{25– 35} (5 μM) injury group, h—Aβ_{25– 35} (10 μM) injury group, i—Aβ_{25– 35} (20 μM) injury group, j—Aβ_{25– 35} (40 μM) injury group

Inverted fluorescence microscopy was used to observe morphological changes of PC12 and SH-SY5Y cells injured by different concentrations of Aβ_{25– 35} (Fig. 8c, d). The PC12 and SH-SY5Y cells in the control group had higher density, fusiform shapes, fuller cell bodies and longer protrusions. As the concentration of Aβ_{25– 35} increased (5, 10, 20, 40 μmol/L), the number of cells in both cell lines gradually decreased, the cell bodies shrank slightly, and the protrusions began to shrink sharply. When the concentration of Aβ_{25– 35} was increased to 40 μmol/L, the protrusions broke significantly, most of the cells contracted sharply, their shape became irregular, and some cells detached and became suspended in the solution.

Therefore, we treated PC12 and SH-SY5Y cells with 20 μM Aβ25-35 for 24 h to establish a nerve injury model.

Neuroprotective Effect of Drug-Loaded Nanoparticles (DZP-CHP)

MTT assays were used to detect the activity of different concentrations of DZP and DZP-CHP (2.5 μM, 5 μM, 10 μM) in PC12 and SH-SY5Y cells [Fig. 9a (i), b(i)]. Tests showed that treatment of PC12 cells with 20 μM Aβ25-35 alone resulted in a significant reduction in cell viability to 48.4 ± 2.8% that in the control group (P  < 0.01). However, after pretreatment with DZP and DZP-CHP (2.5 μM, 5 μM, 10 μM) solutions, the viability of PC12 cells increased significantly. The viability of PC12 cells in the DZP-CHP group was higher than that in the DZP group (P < 0.05). Similarly, treatment of SH-SY5Y cells with 20 μM Aβ_{25– 35} alone resulted in a significant reduction in cell viability to 48.5 ± 4.0% that in the control group (P  < 0.01), while after pretreatment with DZP and DZP-CHP solution (2.5 μM, 5 μM, 10 μM, respectively), the viability of SH-SY5Y cells increased significantly. The viability of SH-SY5Y cells in the DZP-CHP group was higher than that in the DZP group (P < 0.05).

Effect of DZP-CHP on Aβ25-35-injured PC12 and SH-SY5Y cells. een and b The effects of DZP-CHP on the cell survival rate, LDH activity, and mitochondrial membrane potential of PC12 and SH-SY5Y cells injured by Aβ25-35 (#P  < 0.05, ## P  < 0.01 vs Aβ25-35 group; ▲ P < 0.05 vs DZP group). c The effect of DZP-CHP on the apoptosis morphology of PC12 cells injured by Aβ25-35; a—control group, b—Aβ25-35 injury group, c—DZP (5 µM), d—DZP -CHP (5 µM), e—DZP (10 µM), f—DZP-CHP (10 µM). D:the effect of DZP-CHP on the apoptosis morphology of SH-SY5Y cells injured by Aβ25-35; g—control group, h—Aβ25-35 injury group, i—DZP (5 µM), j—DZP-CHP (5 µM), k—DZP (10 µM), l—DZP-CHP (10 µM). E:red/green fluorescence ratio

LDH kits were used to detect the effects of different concentrations of DZP and DZP-CHP (5 μM and 10 μM) on the release of LDH from PC12 and SH-SY5Y cells into the culture medium [Fig. 9a (ii), b (ii)]. Colorimetric measurements showed that the release of LDH from PC12 cells that were exposed to 20 μM Aβ25-35 alone increased significantly by 355.1 ± 16.6% (P  < 0.01). In the presence of DZP and DZP-CHP (5 μM and 10 μM), LDH release from PC12 cells dropped significantly. The effect in the DZP-CHP group was higher than that in the DZP group (P  < 0.01). Similarly, the release of LDH from SH-SY5Y cells exposed to 20 μM Aβ25-35 increased significantly to 357.8 ± 12.5% (P  < 0.01). However, after pretreatment with DZP and DZP-CHP (5 μM and 10 μM), LDH release from SH-SY5Y cells decreased significantly. The effect in the DZP-CHP group was higher than that in the DZP group (P < 0.05).

According to previous reports, depolarization of the MMP leads to loss of Rh123 from mitochondria, which in turn leads to a decline in intracellular fluorescence. Therefore, to characterize changes in the mitochondrial membrane potential in PC12 and SH-SY5Y cells treated with Aβ25-35, DZP, and DZP-CHP (5 μM, 10 μM), rhodamine 123 was used for detection [Fig. 9a (iii), b (iii)]. The results showed that the fluorescence intensity of rhodamine 123 decreased significantly to 44.3 ± 3.8% (P  < 0.01) after incubation of PC12 cells with 20 μM Aβ25-35 for 24 h. However, pretreatment with DZP and DZP-CHP (5 μM, 10 μM) solutions resulted in a significant increase in fluorescence intensity in a dose-dependent manner, and the effect in the DZP-CHP group was higher than that in the DZP group (P < 0.05). Similarly, after treatment of SH-SY5Y cells with 20 μM Aβ25-35 for 24 h, the fluorescence intensity of rhodamine 123 significantly decreased to 42.5 ± 4.6% (P  < 0.01). However, after pretreatment with DZP and DZP-CHP (5 μM, 10 μM), the fluorescence intensity increased significantly, and the effect in the DZP-CHP group was higher than that in the DZP group.

An AO-EB double staining kit was used to detect morphological changes of PC12 and SH-SY5Y cells treated with different concentrations of DZP and DZP-CHP (5 μM and 10 μM) (Fig. 9c, d). After AO-EB double staining, the nuclei of living cells presented green fluorescence under a fluorescence, and the fluorescence of apoptotic cells was orange-red; the higher the degree of apoptosis is, the brighter the fluorescence. Compared with the untreated control group, PC12 and SH-SY5Y cells treated with Aβ25-35 alone showed typical apoptotic characteristics, such as highly condensed and broken nuclei and obvious cell injury. However, pretreatment with DZP and DZP-CHP solution (5 μM and 10 μM) significantly inhibited cell damage and improved cell morphology.