Pembentukan Kompleks Nanopartikel-HSA Berbasis Pullulan dan Pelepasan Obat yang Dipengaruhi oleh Muatan Permukaan

¹ Spektrum HNMR untuk CHP (a), CHSP (b), dan CHAP (c) NP

dimana A _δ2,53 adalah area spektrum di bawah puncak serapan karakteristik metilen (hidrogen), dan A _δ4.74 dan A _δ5.01 adalah area spektrum di bawah puncak serapan karakteristik untuk ikatan glikosidik alfa-1,6 dan alfa-1,4.

Dari ¹ Spektrum H NMR pada CHP, derajat substitusi kolesterol suksinat (CHS) adalah 4,50%. Untuk NP CHSP, gugus metilen (–CH₂ CH₂ –) mencakup dua aspek:suksinat anhidrida dan CHS. Derajat substitusi adalah 12,34% untuk gugus metilen (–CH₂ CH₂ –) dan 7,84% untuk gugus karboksil. Untuk NP CHAP, gugus metilen (–CH₂ CH₂ –) mencakup dua aspek:etilendiamin dan CHS. Derajat substitusi adalah 18,6% untuk gugus metilen (–CH₂ CH₂ –) dan 14,1% untuk gugus amino.

Properti NP CHP, CHSP, dan CHAP

Polimer amfifilik dapat dirakit sendiri dengan dialisis untuk membentuk NP cangkang inti dengan cangkang hidrofilik dan inti hidrofobik, yang dapat diisi dengan obat anti kanker untuk membentuk NP yang mengandung obat. Sifat NP seperti muatan permukaan dan ukuran memainkan pengaruh penting pada kemanjuran pengobatan sebagai pembawa obat [39, 40]. Distribusi ukuran dan potensi zeta NP CHP, CHSP, dan CHAP diukur dengan DLS disajikan pada Gambar. 4. Ukuran rata-rata NP adalah 73,1, 116,9, dan 156,9 nm untuk CHP, CHAP, dan CHSP, masing-masing. Potensi zeta masing-masing adalah 0.698, + 12.9, dan 15.4 mV (Tabel 1).

Potensi zeta (a ) dan distribusi ukuran (b ) dari NP CHP ( ), CHAP NP ( ), dan NP CHSP ( )

Untuk NP pullulan dengan kelompok hidrofobik yang sama, ukurannya lebih besar untuk CHSP bermuatan negatif dan CHAP bermuatan positif daripada NP CHP netral. Dengan demikian, gugus bermuatan mengganggu perilaku perakitan sendiri NP, membentuk NP berukuran lebih besar dengan struktur longgar dalam larutan berair. Potensi zeta dari NP CHP, CHAP, dan CHSP NP masing-masing adalah 0,698 mV, + 12,9 mV, dan 15,4 mV. Dengan demikian, gugus amino dan karboksil dalam polimer dapat membuat NP dengan muatan permukaan yang berbeda dan dengan demikian mengubah sifat permukaan.

Analisis Termodinamika

Analisis termodinamika dilakukan menggunakan ITC dengan titrasi HSA terhadap larutan CHP, CHSP, dan CHAP NP. Saat titrasi HSA ke larutan NP pullulan dengan muatan berbeda, kami menentukan perubahan panas, menyesuaikan sifat sambungan tiga jenis bahan, sambungan, dan jumlah sambungan molekul. Spektrum asli dari termometer titrasi isotermal mencerminkan perubahan panas. Puncak ke atas menunjukkan reaksi pelepasan panas, dan puncak ke bawah menunjukkan reaksi penyerapan panas [41, 42]. Ketika NP CHP, CHAP, dan CHSP dititrasi dengan HSA, panas yang dilepaskan dari kombinasi secara bertahap menurun seiring waktu (Gbr. 5). Secara keseluruhan, 26 tetes larutan HSA dititrasi ke dalam larutan CHP NP, dan puncak spektrumnya naik, menunjukkan sifat reaksi yang eksotermis. Fenomena yang sama diamati ketika larutan HSA dititrasi ke dalam larutan CHSP NP. Namun, ketika larutan HSA dititrasi ke dalam larutan CHAP NP, puncak spektrum untuk empat tetes pertama adalah ke atas, sedangkan dari tetes kelima, puncaknya mengarah ke bawah, menunjukkan reaksi endotermik. Penyerapan HSA NP CHP dan CHSP bersifat eksotermis, sehingga reaksi berlangsung spontan. Penyerapan HSA dari CHAP NP sebagian bersifat endotermik dan sebagian eksotermik, yang mungkin terkait dengan muatan positif CHAP.

Data kalorimetri titrasi isotermal untuk titrasi HSA menjadi a CHP, b CHSP, dan c CHAP NP pada 25 °C. Konsentrasi NP dalam sel (250 L) adalah 12 M dan konsentrasi protein dalam spuit adalah 230 M. Grafik atas menunjukkan data mentah, dan grafik bawah menunjukkan pemanasan terintegrasi

Nilai entalpi mencerminkan panas yang dilepaskan oleh kombinasi HSA dan NP. Perubahan entalpi NP CHP, CHSP, dan CHAP oleh HSA berturut-turut adalah 42,827, 80,3712, dan 22,3951 KJ/mol (Tabel 2). Dikombinasikan dengan perubahan entalpi, reaksi eksotermik, struktur kimia NP amfifilik, dan muatan negatif HSA, interaksi hidrofobik terutama dapat mendorong interaksi HSA dengan NP CHP dan CHSP. Sebagai catatan, dalam titrasi HSA CHAP, panas termasuk nilai negatif. Molekul HSA mengandung kantong hidrofobik yang dapat digabungkan dengan pusat hidrofobik NP [43], sehingga interaksi yang dipicu oleh empat tetes pertama HSA dan CHAP terutama didorong oleh gaya hidrofobik. Meskipun HSA bermuatan negatif dan CHAP bermuatan positif, mulai dari penurunan kelima, ada juga gaya muatan antara HSA dan CHAP karena sifat reaksi endotermik.

Nilai perubahan entropi dapat mencerminkan sulitnya reaksi antara HSA dan NP. Entropi NP HSA dan CHP, CHSP, dan CHAP masing-masing adalah 0,251, 2,775, dan 0,201 KJ/mol K (Tabel 2), sehingga NP CHP dan CHAP lebih mudah berikatan dengan HSA, sedangkan NP CHSP lebih sulit terikat mengikat ke HSA.

Cakupan NP HSA dan CHP, CHSP, dan CHAP masing-masing adalah 1,17, 0,404, dan 0,845. Konsentrasi NP dihitung berdasarkan konsentrasi polimer daripada jumlah partikel. Kami tidak mengetahui berapa banyak partikel polimer tunggal yang mengandung NP tunggal, sehingga kami tidak dapat menentukan secara pasti berapa banyak setiap NP yang teradsorpsi oleh HSA, tetapi berdasarkan nilai cakupan, kami dapat menyimpulkan bahwa NP CHP memiliki adsorpsi HSA tertinggi.

Dalam percobaan sebelumnya, kami menunjukkan bahwa nilai afinitas, K _A , mencerminkan kekuatan gaya pengikatan antara NP dan HSA [32]. Semakin kuat hidrofobisitas NP CHP, semakin kuat afinitas [44]. Konstanta pengikatan HSA dan CHP adalah 27,7 × 10 ⁴ M ⁻¹ , HSA dan CHSP adalah 1,41 × 10 ⁴ M ⁻¹ , dan HSA dan CHAP adalah 412 × 10 ⁴ M ⁻¹ . Jadi, kombinasi HSA dan CHAP bermuatan positif adalah yang terkuat, diikuti oleh CHP bermuatan netral dan CHSP bermuatan negatif. Kombinasi terkuat dari HSA dan CHAP dapat dikaitkan dengan gaya hidrofobik, gaya tarik-menarik timbal balik dari gaya muatan dan mungkin muatan yang saling eksklusif antara HSA dan CHSP.

Spektroskopi Fluoresensi

Dengan spektrum fluoresensi, kami mempelajari interaksi antara HSA dan tiga NP dengan muatan permukaan yang berbeda. HSA mengandung 585 residu asam amino, dengan hanya satu residu Trp pada 214 (Trp214), dan spektrum fluoresensinya mendominasi di wilayah UV. Ketika molekul lain berinteraksi dengan HSA, spektrum fluoresensi Trp dapat berubah, tergantung pada interaksi antara HSA dan molekul lain. Ketika ketiga NP dicampur dengan HSA, puncak emisi maksimum spektrum fluoresensi HSA tidak mengalami pergeseran kimia; hanya intensitasnya yang melemah sampai batas tertentu (Gbr. 6A). Puncak emisi maksimum HSA diamati ketika HSA dikombinasikan dengan NP CHAP.

A Spektrum fluoresensi untuk HSA (1,5 × 10 ^− 5 mol/L) tanpa (a) dan dengan (b) CHSP, (c) CHP dan (d) CHAP NP dengan konsentrasi yang sama (4.2 × 10 ^− 6 perempuan jalang). B Intensitas emisi HSA dengan CHSP, CHP, dan NP CHAP pada 343 nm dari waktu ke waktu

Studi eksperimental menunjukkan bahwa menggabungkan NP HSA dan pullulan menunjukkan proses yang kompleks [45]. Kami menambahkan tiga NP ke dalam larutan HSA dan menemukan bahwa intensitas fluoresensi dari tiga kompleks NP-HSA menurun secara bertahap (Gbr. 6B). Kesetimbangan tercapai untuk HSA-CHSP pada 556,3 nm setelah 12 jam, untuk CHP-HSA pada 534,3 nm setelah 10 jam, dan untuk CHAP-HSA pada 512,3 nm setelah 8 jam. Waktu yang diperlukan ketika intensitas fluoresensi kompleks NP-HSA yang berbeda seimbang terkait dengan muatan yang dibawa oleh NP. Waktu terlama yang diperlukan untuk mencapai kesetimbangan diamati pada CHSP–HSA bermuatan negatif, diikuti oleh CHP–HSA yang tidak bermuatan.

Pengurangan yang cepat dalam intensitas fluoresensi awal dari tiga kompleks NP-HSA (ditunjukkan pada Gambar. 6A) adalah karena adsorpsi yang cepat dari NP dan HSA. Intensitas fluoresensi berikutnya menunjukkan penurunan lambat ke status konstan karena interaksi NP dengan HSA adalah proses kombinasi yang lambat. Intensitas fluoresensi konstan mencerminkan status saturasi kompleksasi. Interaksi antara tiga NP bermuatan berbeda dan HSA menjalani proses rekombinasi cepat awal dan proses rekombinasi lambat kemudian. Kombinasi HSA yang bermuatan negatif dengan CHSP yang bermuatan negatif membutuhkan waktu yang lebih lama untuk mencapai saturasi kompleks dibandingkan dengan CHP yang tidak bermuatan. HSA bermuatan negatif yang digabungkan dengan CHAP bermuatan positif membutuhkan waktu tersingkat untuk mencapai kejenuhan kompleks.

Gambar 7A–C menunjukkan spektrum HSA yang dikombinasikan dengan konsentrasi CHSP, CHP, dan CHAP NP yang berbeda untuk membentuk kompleks NP-HSA. Dengan meningkatnya konsentrasi NP, puncak penyerapan maksimum kompleks NP-HSA menurun menunjukkan korelasi terbalik.

Spektrum fluoresensi HSA (1,5 × 10 ^− 5 mol/L) dengan CHSP (A ), CHP (B ), dan CHAP (C ) pada konsentrasi yang berbeda (a) 0, (b) 2.07 × 10 ⁻⁷ , (c) 3,31 × 10 ⁻⁷ , (d) 4.14 × 10 ⁻⁷ , (e) 8.28 × 10 ⁻⁷ , (f) 20.7 × 10 ⁻⁷ , (g) 33,1 × 10 ⁻⁷ , dan (h) 41,4 × 10 ⁻⁷ perempuan jalang. D Plot (n = 7) untuk F ₀ /(B ₀ F) vs 1/[Q ], T adalah konsentrasi CHSP (–◆–), CHP (–■–) dan CHAP (–▲–), masing-masing

CHAP-HSA bermuatan positif menunjukkan waktu tersingkat untuk mencapai kesetimbangan.

Kami menggunakan persamaan Stern–Volmer yang dimodifikasi untuk menganalisis data pendinginan fluoresensi:

dimana f _a adalah fraksi kontak zat fluoresen dengan quencher, K _q adalah konstanta pendinginan Stern–Volmer, F ₀ adalah intensitas fluoresensi pada 342 nm tanpa quencher, F adalah intensitas fluoresensi pada 342 nm dengan quencher, dan [Q ] adalah konsentrasi quencher.

Dari gambar fungsional F ₀ /(B ₀ B ) pasangkan 1/[Q ], kita dapat memperoleh nilai f _a dan K _q dari lereng dan intersep (Gbr. 7D). Assuming that the observed fluorescence intensity changes mainly due to the interaction between NPs and HSA, the quenching constant can be seen as a binding constant for the formation of complexes. The binding constants for HSA and CHSP, CHP, and CHAP molecules were 2.02, 2.99, 4.72 × 10 ⁵ M ⁻¹ , masing-masing. In the previous study, we discussed the interaction between HSA and CHP NPs with different hydrophobicity substitutions [30]. The hydrophobic interaction between HSA molecules and CHP cholesterol played an important role in the formation of CHP–HSA. The greater the hydrophobic substitution of CHP, the greater the binding constant of CHP and HSA.

In the present study, we found that the value of the binding constant (K _b ) is related to the electrical properties of the NP. A surface with a positive charge, such as CHAP, has the largest K _b , and a surface without a charge, such as CHP, has the second largest K _b . The K _b for CHSP, with a negative charge, was the lowest. Therefore, in addition to the hydrophobic interaction between NPs and HSA, the electrostatic interaction between them also plays an essential part in the formation of NP–HSA complexes.

In addition, the f _a values for CHSP, CHP, and CHAP NPs were 0.269, 0.288, 0.38, respectively, so part of the Trp residue was involved in this reaction. Furthermore, the amount of HSA was too much at a given NP/HSA concentration, so free HSA molecules presented in the reaction system.

CD Spectrum Analysis

Figure 8A shows the CD spectra (a) free HSA, (b) CHSP–HSA, (c) CHAP–HSA, and (d) CHP–HSA in solution at 25 °C. The samples were complex I. There are two negative bands at 208 and 222 nm of the UV region in HSA spectra, which are the characteristic peaks of the α -helical structure. The α -helical content of free HSA was 55%. At the beginning of the complex, with the recombination of HSA and CHSP, CHAP, and CHP NPs, the α -helical content of HSA was reduced to 52.0%, 48.57%, and 48.0%, respectively.

A CD spectra for (a) free HSA, (b) CHSP–HSA, (c) CHAP–HSA, and (d) CHP–HSA in solution at 25 °C. The samples were complex I. B CD spectra for (a) free HSA, (b) CHSP–HSA, (c) CHAP–HSA, and (d) CHP–HSA in solution at 25 °C. The samples were complex II. C Ellipticity at 208 nm for HSA interacting with CHSP (–▲–), CHAP (–●–) and CHP (–■–) over time

Figure 8C shows that the ellipticity changes of the three samples at 208 nm over time. The ellipticity of HSA combined with CHAP and CHP NPs increased from 0 to 12 h and leveled off at 12 h, but that of HSA with CHSP NPs increased faster, i.e., the ellipticity increased from 0 to 9 h and leveled off at 9 h. Therefore, the ellipticity of the three samples gradually increased over time and the α-helical content gradually decreased; when the ellipticity maintained constant, the recombination of the sample and HSA was completed.

Figure 8C illustrates that the fastest surface absorption rate was presented on the combination of CHSP and HSA. The size, charge, and hydrophobicity of NPs can influence the migration rate of HSA to the center of NPs. The surface of CHP is not charged, the surface of CHSP is negatively charged, and the surface of CHAP is positively charged. Owing to the presence of the negative-charge mutual exclusion between CHSP and HSA, the resistance of HSA migrating to the CHSP NP center becomes larger. The traction of NPs on HSA is driven by the hydrophobic interaction forces. The traction of the three NPs on HSA is identical, so the lowest velocity of HSA migrating to the center of NPs was observed on the combination of HSA and CHSP NPs. Because the particle size is in the order of CHSP> CHAP> CHP, the particle density displays a reverse order, i.e., CHSP

The ITC results show that the amount of HSA migrating toward the center of CHSP NPs is the smallest but with the fastest speed compared with other types of NPs. Table 3 shows that the α-helix content of HSA migrating toward the center of CHP NPs was the lowest. The more the secondary structure of HSA was damaged, the faster the HSA migrated toward the center. The fastest speed was observed on HSA migrating to the center of CHSP NPs (Fig. 8C).

Figure 8B shows CD spectra for (a) free HSA, (b) CHSP–HSA, (c) CHAP–HSA, and (d) CHP–HSA in solution at 25 °C. The samples were complex II. After the complexation is completed, the α -helical content of HSA recombined with CHSP, CHAP, and CHP was reduced to 46.27%, 44.55%, and 42.91%, respectively (Table 3). With the increase in interaction time, the secondary structure of HSA was changed and the α -helical content was reduced during the process of complexation with NPs.

Drug Release

We measured the drug release rate of MTO with the three kinds of drug-loaded NPs and drug-loaded NP–HSA complexes. The drug release for free MTO was about 99.8% at 4 h (Fig. 9). The release rate in 48 h for CHP, CHAP, and CHSP NPs was 53.68%, 58.54%, and 63.24%, respectively. The drug release from all NPs was fast in the first 8 h, which was a burst release process, and the drug release remained stable after 12 h, which was a sustained release process. In vitro drug release from NPs is not affected by gastrointestinal pH and enzymes, and the dissolution of nanomaterials results in little drug release. The release is mainly determined by dissolution and diffusion [46]. The 48-h drug release rate was in the order of CHSP> CHAP> CHP, corresponding to the size of NPs. The fastest release rate was found on CHSP, which was negatively charged with the largest size. The second fast drug release rate was found on which was positively charged with the size smaller than CHSP. CHP was electrically neutral, and its drug release rate was minimal. Hence, the polymer surface groups involved in the formation of NPs affect the size of NPs and ultimately the drug release of NPs.

Mitoxantrone (MTO) release of pullulan NPs in phosphate-buffered saline (PBS) at 37 °C in vitro (□:free mitoxantrone, ○:CHP, △:CHAP, ▽:CHSP, ◁:CHAP–HSA,◇:CHP–HSA, ▷:CHSP–HSA)

The drug release rate from the combinations of HSA and CHAP, CHP, and CHSP in 48 h was 32.45%, 33.86%, and 35.76%, respectively. After the combination of NPs and HSA, the drug release in 48 h was significantly decreased as compared with HSA-free NPs, which was mainly attributed to the resistive effect and adsorption effect of HSA. At 48 h, the drug release of the compounds was in the order of CHSP–HSA> CHP–HSA> CHAP–HSA, while the drug release of HSA-free NPs was in the order of CHSP> CHAP> CHP. Although NP–HSA compounds showed significantly slow drug release, the total release of CHP-HSA decreased by 21.23% in 48 h, whereas the total release of CHAP-HSA decreased by 25.68% and that of CHSP-HSA by 28.48%. The drug release of the NPs is related to the size of the NPs and the polymer hydrophobic groups on NP surface. The adsorption of HSA can lead to significant slowdown in drug release, which is related to the hydrophobicity of NPs and also to the surface charge of NPs [46]. The adsorption of HSA is closely related to the size of the NPs and the degree of substitution of the hydrophobic groups of the polymer during the self-assembly process. Nevertheless, the drug release of the NPs is ultimately determined by the properties of the NP itself.

Discussion

As shown in Fig. 10, the formation of the NP–HSA complex is driven by a hydrophobic force between cholesterol groups of the particle core and the aromatic amino acid of the hydrophobic domain of HSA. After mixing, HSA interacts with the surface cholesterol unit and is rapidly adsorbed to the NP surface. Then, the adsorbed HSA on the NP surface is processed because of the hydrophobic forces derived from the cholesteric unit in the particle core. When overcoming the steric hindrance of polysaccharide chains in the NP shell, the adsorbed HSA gradually migrates to the core. After the hydrophobic interaction and resistance of the hydrophilic polysaccharide chain are balanced, the HSA molecule enters the particle core to become hydrophobically bound to cholesterol groups to form the NP–HSA complex.

Adsorption of HSA to NPs

For CHAP and CHSP NPs, the recombination of HSA is a complex process also subjected to the charge interaction with HSA under the traction of the hydrophobic driving force. The binding constants of the three kinds of NPs with the same hydrophobic substitution and different surface charge were in the order of CHAP> CHP> CHSP. The electrical properties also play a major part in the formation of NP–HSA. In this process, the formation of the CHSP–HSA complex was blocked by the structure of the NP shell and the repulsive force between the negative charges, which led to their loose connection. During the rapid adsorption and slow recombination, the degree of spiraling of HSA is lower for CHSP NPs than CHP NPs and CHAP NPs. Therefore, the surface charge of NPs not only changes the nature of the particles themselves but also affects the protein complex.

In the current study, we investigated the effect of NP surface charge on the interaction between NPs and proteins (Fig. 10). Three different charges of pullulan NPs with HSA adsorption still showed rapid adsorption and slow recombination. The number of HSA molecules with positively charged CHAP complex was the most, including rapid adsorption of NP–HSA by hydrophobic forces, HSA molecule migration to the center, and the HSA molecules adsorbed on the surface of NPs by charge action. CHP- and CHSP-adsorbed HSA molecules were mainly distributed in the hydrophobic center of NPs, with CHSP adsorbing fewer HSA molecules. The adsorbed number of HSA molecules is related to the hydrophobicity of NPs. The greater the degree of substitution of hydrophobicity, the more HSA is adsorbed [41]. The cholesterol substitutions of the three NPs were the same, and the number of HSA molecules adsorbed by positively charged NPs was the highest, so the adsorption of NPs and HSA was related to the hydrophobicity and surface charge of the NPs.

The surface adsorption capacity between NPs and HSA is also related to the hydrophobicity and charge of NPs. The binding force between HSA and NPs is determined by the hydrophobicity, surface charge, size, and structure of NPs. The α-helicity was decreased most at the beginning of adsorption and the complete CHP–HSA complex. CHP NP has the smallest size and highest density. The CHP NPs migrated toward the center by the hydrophobic traction; the sugar chain of the CHP NP shell was larger to inhibit the migration toward the center. The extension of the peptide chain of HSA is larger, with the α -helix decreased the most. Although CHAP NPs have hydrophobic and charge forces, they possess relatively large size, loose structure, small resistance in the periphery, small extension of the peptide chain, and small content of the α -helix. Some HSAs remained on the surface of NPs through the charge force of adsorbing, and the α -helical content is also smaller in this part of the HSA. The α -helix content of CHAP decreased less than that of CHP, mainly due to the peptide chain extension-induced central pulling force which led to α-helix content decline. During the process of the CHSP and HSA complexation, the role of the central pulling force has a reverse direction of the charge force, thereby resulting in weakening the center of the migration force. CHSP NPs are larger than CHAP NPs, and the structure of CHAP NPs is loose. Because the adsorbed number of HSA on CHAP is higher than that on CHSP, the decrease of α -helicity in CHSP is less than that in CHAP NPs. Therefore, the interaction between NPs and HSA and the decrease in α -helicity are all related to the size, density, hydrophobicity of substitution, surface charge of the NPs, and number of HSA connections.

After the NPs enter into the blood, protein adsorption affects the functions of NPs, such as the slow and controlled drug release, the travel from the blood circulation passing through the vascular barrier, targeting tissue, and entering cells. NPs interact with the HSA in the body and affect the in vivo behavior of NPs. The number of adsorbed proteins is closely related to the properties of the NPs. HSA adsorbs NPs, which affects the distribution in organs and removal of NPs, thereby altering the concentration of the drug in the body and the efficacy of the drug.

Finally, the properties of NPs, such as size, hydrophobicity, and surface charge, affect the drug release of NPs in vivo. We can design specific materials to perform specific functions with specific protein adsorption.