Preparation of folic acid-conjugated albumin nanoparticles 1 containing paclitaxel using high-pressure homogenisation 2 coagulation method

In this study, we prepared and evaluated folic acid-conjugated albumin-paclitaxel (FA-BSA-PTX) nanoparticles using a new green technique, called the high-pressure 11 homogenisation coagulation method (HPHCM). The effect of process parameters 12 such as BSA concentration, coagulant concentration, homogenisation time, 13 homogenisation pressure, water/ethanol ratio, and BSA/PTX ratio was analysed to 14 optimise nanoparticle size, albumin conversion rate, and encapsulation efficiency. BSA 15 concentration was found to exert a great influence on albumin conversion rate and 16 particle size. Meanwhile, the BSA/PTX ratio significantly affected the nanoparticle 17 encapsulation efficiency. Electron microscopy showed that the freeze-dried particles 18 mostly existed in the form of dimers and trimers with an average particle size of 300– 19 400 nm. Infrared spectroscopy indicated that PTX was well encapsulated in BSA. 20 Raman spectra of the synthesised nanoparticles indicated changes in the disulphide 21 bond configuration and protein structure. In vitro drug-release analysis showed that 22 crosslinked nanoparticles exhibited a sustained release. Furthermore, in vitro cell- 23 uptake studies on HeLa cells showed that FA can be used as a targeting ligand for 24 albumin carriers to enhance the active targeting effect of the nanoparticles with a high 25 FR expression. These results suggest that HPHCM is an effective method to prepare 26 FA-BSA-PTX drug-delivery systems.


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Nanoparticle-based drug-delivery systems are considered to show great potential 31 for cancer treatment as these carrier systems exhibit high drug absorption, adjustable 32 drug-release rate, and targeted delivery, especially in the case of hydrophobic drugs binding capacity for drugs with different physical and chemical properties. In addition, 46 the BSA molecule contains functional groups such as amino and carboxyl groups 47 owing to which albumin nanoparticle carrier surfaces can be easily modified by ligands 48 [8,9]. 49 The main purpose of targeted cancer therapy is to deliver drugs to tumour cells in 50 order to localise high drug concentrations at tumour sites and reduce side effects 51 [10,11]. Passive targeting is inherent in drug-loaded nanoparticles. Meanwhile, to 52 achieve an active targeting function, ligand modification may be adopted [12]. Folic 53 acid (FA), a common targeting ligand, is a low molecular weight vitamin whose folate 54 receptor (FR) is overexpressed on the surfaces of a variety of cancer cells such as 55 breast, ovary, lung, kidney, colon, and brain cancer cells; however, its expression is 56 minimal in healthy tissues and organs. The FR expression level in cancer cells is 100-57 4 300 times higher than that in normal tissues [13]. Therefore, targeted delivery can be 58 achieved by FA-linked nanoparticles entering the cytoplasm via FR mediation and 59 endocytosis. FR is extensively studied for cancer diagnosis and treatment [14] due to 60 several key characteristics. Firstly, FA can be easily synthesised, is inexpensive, and 61 is easy to chemically modify and characterise [15]. Secondly, FA itself has no 62 immunogenicity and exhibits high structural stability even after chemical modification, 63 thus maintaining a high affinity with FR [16]. 64 Traditionally, albumin nanoparticles are prepared by chemical crosslinking, thermal 65 denaturation, or desolvation. Chemical crosslinking is non-specific and depends only 66 on the reactivity of the nucleophilic groups (such as amino and hydroxyl groups) in the 67 protein structure. Nevertheless, the crosslinking agents used in this method, such as 68 formaldehyde, exhibit high levels of toxicity and result in residues [17,18]. Thermal 69 denaturation, by techniques such as solution thermal crosslinking or spray drying, 70 irreversibly alters the protein structure and the resultant protein nanoparticles exhibit 71 poor biodegradability and a wide size distribution [19]. Using the desolvation method,   where Wfree is the amount of free drug and Wtotal is the amount of total drug. where Wfree is the amount of free albumin and Wtotal is the amount of total albumin.  The samples were scanned in the 2θ range of 3° to 90° at 0.02°/min.  factors into account, in this study, the effect of different coagulants on the particle size 266 and conversion rate was investigated.

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As shown in Fig. 2 coagulant, owing to which the nanoparticles could not be effectively dispersed.

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The particle size of albumin nanoparticles in the 0.5 mg/mL coagulant group was 278 smaller than that in the 1.25 mg/mL group. Particle-size order in the 0.5 mg/mL 279 coagulant group was magnesium chloride < calcium chloride < calcium lactate < 280 gluconolactone, with nanoparticles prepared using magnesium chloride being the 281 smallest (310.6 nm). In the 1.25 mg/mL coagulant group, the particle-size order was 282 magnesium chloride < calcium chloride < calcium lactate < gluconolactone, with 283 nanoparticles prepared using magnesium chloride being the smallest (398.6 nm).

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Thus, considering the particle-size and conversion-rate indicators, magnesium 285 chloride was selected as the optimal coagulant and used in the rest of the study.  (Table 1) on the quality of the produced nanoparticles.

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The effect of BSA concentration on the synthesised albumin nanoparticles is 293 illustrated in Fig. 3a. As the BSA concentration increased from 5 to 40 mg/mL, particle 294 size gradually increased from 327 to 377 nm. The change in particle size can be 295 explained using the following relationships: nucleation rate, which in turn increased the particle size and conversion rate.

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The effect of water/ethanol volume ratio on albumin nanoparticles is shown in Fig.   331 3c. As the water/ethanol volume ratio decreased from 30 to 10, the particle size initially 332 14 decreased and then increased. The minimum particle size of 323 nm was observed at 333 water/ethanol = 25/1, which is consistent with previously reported results [31]. 334 Meanwhile, the BSA conversion rate decreased when the water/ethanol volume ratio 335 decreased from 30 to 10. The encapsulation rate initially increased and then 336 decreased and the highest value (92.2%) was observed at water/ethanol = 20/1.

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The effect of homogenisation pressure on the properties of BSA nanoparticles is 338 shown in Fig. 3d. As the pressure increased from 600 to 1000 bar, particle size initially . In this process, the conversion rate also increased.

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Statistically, the influence of homogenisation pressure on conversion rate was highly 348 significant (p < 0.01). The encapsulation rate initially increased and then decreased, 349 reaching the highest value (89.7%) at a homogenisation pressure of 700 bar.

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The effect of homogenisation time on albumin nanoparticles is illustrated in Fig. 3e.

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When the homogenisation time increased from 3 to 9 min, the particle size increased strongly attracted to each other, resulting in a sudden increase in particle size.

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Statistically, the influence of homogenisation time on particle size was found to be 363 significant (p < 0.05). Furthermore, the conversion rate increased as the 364 homogenisation time increased. Meanwhile, the encapsulation rate increased initially 365 and then decreased, with the maximum value (91.4%) observed at 12 min.

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The effect of the BSA/PTX ratio on albumin nanoparticles is shown in Fig. 3f. When 367 the BSA/PTX ratio decreased from 25 to 5 (dosage increases), the conversion rate  The morphology of BSA-PTX nanoparticles were analysed using TEM and SEM, 380 as shown in Fig. 4. It can be seen in the SEM images (Fig. 4a) that albumin is in the 381 form of irregular flakes while raw PTX is in the form of crystalline strips (Fig. 4b). It can 382 also be concluded from the SEM images ( Fig. 4c and d) of BSA-PTX nanoparticles 383 that the freeze-dried particles were mostly in the form of dimers and trimers with an 384 average particle size of 300 nm; in terms of shape, most single particles were spherical.

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The TEM image of BSA-PTX nanoparticles (Fig. 4f) shows that the freeze-dried Raman spectroscopy is a rapid, simple, repeatable, and non-destructive qualitative 420 and quantitative technique that does not require special sample preparation. Thus, we 421 employed this method to analyse the BSA-based nanoparticles prepared in this study.

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As shown in Fig. 6, peaks were observed at 1655 and 1339 cm -1 in the spectrum    fluorescence was absent in the case of FITC (Fig. 10C) also be studied to develop efficient drug carriers for targeted delivery.