Laetitia Théron
email: laetitia.theron@inrae.fr

Thierry Sayd

Christophe Chambon

Antoine Vautier

Claude Ferreira

Laurent Aubry

Annie Vénien

Didier Viala

Thierry Astruc

Vincenza Ferraro

Véronique Santé-Lhoutellier

Meat and Muscle Biology™

Keywords: PSE-like, ham, proteomic, imaging, mass spectrometry, predictive model

Pale Soft Exudative (PSE)-like muscle defect is of great importance in the cooked ham industry because of the economic losses it can cause. The flagship product is the "Jambon supérieur," a polyphosphate-free cooked ham, usually sold sliced and packaged. Slicing is an automatic process that reveals the defect as holes in the slice leading to slicing losses. Up to now, the PSE-like defect has only been detected on raw meat after deboning the pork leg because it affects the inner part of the semimembranosus muscles and also the adductor muscles. The objective of this study was to develop innovative approaches that combine mechanistic elucidation and the discovery of potential biomarkers (i) at the level of the muscle and (ii) at the level of the live animal by analyzing proteins from plasma. The use of chemometrics for the spectral fingerprinting of pig plasma was chosen to predict the PSE-like muscle defect in raw hams.

Introduction

The Pale Soft Exudative (PSE)-like defect that occurs in raw meat used to produce cooked hams is a major issue. Indeed, it can represent up to 50% of the total processed ham, the French "jambon supérieur" cooked ham, and leads to significant economic losses [START_REF] Vautier | Prediction level of meat quality criteria on "PSE-like zones" defect of pork's ham. 54th International Congress of Meat Science And Technology (ICoMST), 7A-27, Cape Town, South Africa[END_REF]. The defect is mainly detected during the automated slicing process, by the appearance of holes within the ham slices. Because of the histological and biochemical similarities observed on muscles, this defect is compared to PSE meat, but in most cases, it is located in the deepest regions of the semimembranosus muscle, near the femur bone [START_REF] Vautier | Prediction level of meat quality criteria on "PSE-like zones" defect of pork's ham. 54th International Congress of Meat Science And Technology (ICoMST), 7A-27, Cape Town, South Africa[END_REF]. The defect finds its origin in a combination of risk factors that are associated with more than just stress or stress sensitivity, and chilling was not found to influence the defect frequency [START_REF] Vautier | Prediction level of meat quality criteria on "PSE-like zones" defect of pork's ham. 54th International Congress of Meat Science And Technology (ICoMST), 7A-27, Cape Town, South Africa[END_REF].

Although previous observations have suggested a gradient within the ham [START_REF] Franck | Observations préliminaires sur le jambon déstructuré-Description du phénomène et étude de quelques facteurs de variation[END_REF] (indeed, the defect starts to appear on the internal surface of the semimembranosus and the adductor, then spreads out toward subcutaneous regions for strong cases without being noticeable from the outside of bone-in hams), no information is available regarding the progression of the PSE-like defect within muscles. Furthermore, because of its high variability, there is a strong need for a discriminative sort key to classify raw material prior to ham processing. Recently, the use of near-infrared spectroscopy was proven efficient to classify fresh pork meat and to predict the suitability of fresh pork meat for the production of cooked ham [START_REF] Neyrinck | Application of near-infrared spectroscopy for the classification of fresh pork quality in cooked ham production[END_REF].

In this context, we first aimed to elucidate the development of the PSE-like defect by its characterization from the tissue to the molecular scale using mass spectrometry approaches. In the second part, we developed a predictive method to classify raw material prior to ham processing using blood samples, using the spectral fingerprints of plasma proteins studied by Matrix-Assisted Laser Dissociation Ionization Time-of-Flight (MALDI-TOF) mass spectrometry and Attenuated Total Reflectance-Fourier Transform InfraRed (ATR-FTIR) Spectroscopy.

Materials and Methods

Animal, muscle, and blood samples

A batch of 120 pigs (from a sow Large White × Landrace and Pietrain sire), from different farms, were slaughtered at the commercial abattoir "la Guerche de Bretagne, France." Carcasses were blast-chilled for 1 h and 15 min, and blood was collected from each animal and treated as described in the following section. At 24 h post mortem, after pH and temperature stabilization, hams were sorted according to their level of destructuration on a scale from 1 to 4 [START_REF] Vautier | Prediction level of meat quality criteria on "PSE-like zones" defect of pork's ham. 54th International Congress of Meat Science And Technology (ICoMST), 7A-27, Cape Town, South Africa[END_REF], their pH, temperature, and exudate values. From the 120 animals, 20 were selected, based on the destructuration score-10 "normal hams" (NH) and 10 "PSE-like hams" (DH). The inner part (DHi) and outer parts (DHo) of semimembranosus muscles were sampled in each of the DH, and the inner part of semimembranosus muscles was sampled in each of the NH.

MALDI-TOF mass spectrometry imaging of ham samples

At 24 h post mortem, semimembranosus muscle samples (1 × 1 × 1 cm) were collected, positioned on a cork plate, and cryofixed by immersion at -160°C in isopentane cooled with liquid nitrogen (-196°C). Serial cross-sections (10 μm thick) were cut using a cryostat (Microm HM 560; Thermo Scientific) and were collected on glass slides for histological stains and on indium tin oxide glass slides (Bruker Daltonik, Bremen, Germany) for MALDI-mass spectrometry imaging (MSI). The glass slides were stored under vacuum until use. The muscle sections were subjected to 2 washing steps at 70% and 95% ethanol to deplete lipids and then dried in a desiccator for 30 min. The sinapinic acid matrix was 10 mg/mL in water/acetonitrile at 60:40 (v/v) with 0.2% trifluoroacetic acid, applied using ImagePrep (Bruker Daltonics) according to the manufacturer's recommendations. The spectral data were acquired on an Autoflex Speed MALDI-TOF/TOF mass spectrometer equipped with a Smartbeam laser, using FlexControl 3.4 and FlexImaging 3.0 software packages (Bruker Daltonics). Ions were detected in positive linear mode over a mass range of m/z 2,000-20,000 with a sampling rate of 0.63 GS/s. The lateral spatial resolution was set to 75 μm, and a total of 500 laser shots were accumulated per pixel at constant laser power, using random movement within each pixel. The deflection was set at an m/z of 1,500 and laser focus at medium. Analyses were performed using a detector gain of 2.69 V, ion source voltage 1 at 19.5 kV, ion source voltage 2 at 18.15 kV, and lens voltage at 7 kV. Spectral data were loaded into SCiLS Lab 2016 software (http://scils.de/; Bremen, Germany). The following workflow was used for data treatment: baseline subtraction using the TopHat algorithm, normalization by the total ion current algorithm, and peak picking using the orthogonal matching pursuit algorithm, with peak alignment and spatial denoising.

Plasma proteins spectral fingerprints by MALDI-TOF mass spectrometry and FTIR spectroscopy

Immediately after slaughter, a whole blood sample was collected from each pig in a heparinized tube (Venosafe, Terumo, Shibuya, Tokyo, Japan). After 5 stirrings, the tubes were centrifuged at 1500g for 10 min. The plasma was recovered and frozen immediately in liquid nitrogen. The samples were stored at -80°C until use. For the MALDI-TOF mass spectrometry protein fingerprint, plasma proteins were prepurified using Spin Tubes (C18 Agilent Peptide Cleanup, Agilent Technologies, Wilmington, DE) according to the manufacturer's instructions. Then, 1 μl of pre-purified plasma proteins was manually spotted in triplicate on a polished steel target (MTP 384 Target Plate Polished Steel, Bruker Daltonics GmbH, Bremen, Germany) at a ratio of 1:1 with the matrix. The matrix used was α-cyano-4-hydroxycinnamic acid matrix at 7 mg/mL in water/acetonitrile 50:50 (v/v) with 0.2% trifluoroacetic acid. The mass spectrometer used to acquire the protein fingerprinting was an Autoflex Speed MALDI-TOF/TOF with a Smartbeam laser, using FlexControl (version 3.4) software (Bruker Daltonics GmbH, Bremen, Germany). A total of 4,000 spectra were accumulated randomly per sample. The laser power was constant for all the samples, and the laser focus was set at medium. Ion detection was done in linear mode at a mass range of m/z 1,000-10,000, with a sampling rate of 0.31 GS/s. Acquisition deflection was set at m/z 1,000. The detector gain was set at 2,500 V, the ion source voltage 1 at 19.56 kV, the ion source voltage 2 at 18.11 kV, and the lens voltage at 7 kV. External calibration of spectra was done through the deposition of a protein standard (Protein Calibration Standard I, Bruker Daltonics) before each measurement on the same target. MALDI-TOF spectra were processed using FlexAnalysis software (version 3.4) (Bruker Daltonics GmbH, Bremen, Germany). Baseline subtraction using the TopHat algorithm was set with a 5% minimal baseline width.

Smoothing was performed with a 1 Da width and 5 cycles. Individual spectra were normalized using the Total Ion Count (TIC) value. Peak picking was applied using the peak intensities with a signal to noise ratio of 2. Infrared spectra were obtained using a Bruker Tensor II spectrometer, and an ATR accessory with 250 μm × 250 μm diamond crystal (Specac Ltd., Orpington, UK) operated by the OPUS 7.5 software (Bruker, Bremen, Germany). The spectral resolution was set at 4 cm -1 , and 100 scans were used for each measurement in the range of 4,000-600 cm -1 . All the sample acquisitions were performed 5 times as follows: 1 μL of plasma was spotted on the diamond crystal and left to dry at room temperature for 8 min. Between samples, the ATR crystal was cleaned with deionized water and ethanol, and for each sample measurement, a background spectrum was recorded for correction. The 3 best spectra out of 5 were analyzed. After atmospheric compensation and baseline correction, the spectra were pre-processed by the second derivative operation and cropped to obtain 2 areas of interest: 1,800-900 cm -1 and 3,450-2,700 cm -1 . Each area was then normalized using vector normalization, and the mean spectra of the 3 replicates were used.

Chemometrics

Neuronal network analysis was performed using Orange software [START_REF] Demsar | Orange: Data mining toolbox in Python[END_REF]. The number of neurons per hidden layer was set at 100 with an activation function by logistics. The solver parameter for weight optimization was "Adam," a stochastic gradient-based optimizer. The alpha parameter, the regularization term, was set at 0.0001, and the maximum number of iterations was set at 200. Internal validation was performed using a stratified 5-fold cross-validation. The classification results using the predictive model are given for each sample of PSE-like muscle defect (DH, n = 6) and normal (NH, n = 7) hams. The classification assignment is given (NH/ DH) and shown in gray in the case of misclassification. The score probability of classification assignment is given for each class, i.e., DH and NH.

Results

Spatial variability of the DH defect

The spatial variability of the PSE-like defect that occurs in cooked hams was investigated by MSI by MALDI-TOF. To determine whether the defect progressed internally within the ham, indicating a local heterogeneity, the relationship between NH molecular maps with both DHi and DHo was studied. The training set used to build the predictive model was composed of the spectral data of the DHi and DHo. Then, the data from the NH were classified using this model to determine the class to which they were closer: DHi or DHo. The model's performance was evaluated by crossvalidation accuracy, which was 87%. The model's performance can be expressed as its capability to describe The evaluation results of the predictive model are given for each spectral method, i.e., MALDI-MS and IR, and are expressed as AUC, CA, F-1, Precision, and Recall values (from 0 to 1). AUC is the Area under ROC, the Receiver-Operating Curve; CA is the Classification Accuracy, the proportion of correctly classified samples; F-1 is a weighted harmonic mean of precision and recall; Precision is the proportion of true positives among instances classified as positive, e.g., PSElike hams correctly identified as PSE-like hams; and Recall is the proportion of true positives among all positive instances in the data, e.g., the number of PSE-like hams among the normal hams. The classification results using the predictive model are given for each individual sample of PSE-like muscle defect ("PSE," n = 6) and normal ("N," n = 7) hams. The classification assignment is given (N or PSE), shown in gray in the case of misclassification. The score probability of classification assignment is given for each class, i.e., PSE and N. This result confirmed the hypothesis according to which the structural defect is internal, and that the DHo present more similarities to the NH. Boxplots and all the individual intensities measured on each pixel are shown in Figure 1B, as well as an example of an ionic map for each ion. It appeared very clear that these two ions are co-localized and that their intensities are correlated. The mass shift of 27 Da could be due to oxidation of arginine (to glutamic acid) (https://abrf. org/delta-mass), and the oxidation product of arginine may be glutamic semialdehyde, a carbonyl that is a very well described modification in meat products [START_REF] Estévez | Protein carbonyls in meat systems: A review[END_REF]. Indeed, the carbonylation of proteins from chicken breast meat has been studied and linked to PSE meat [START_REF] Estévez | Oxidative damages to poultry: From farm to fork[END_REF][START_REF] Carvalho | Underlying connections between the redox system imbalance, protein oxidation and impaired quality traits in pale, soft exudative (PSE) poultry meat[END_REF]. Despite the absence of direct protein identification using MSI by MALDI-TOF, altogether these results could suggest that m/z 4128.7 and m/z 4155.6 are the same protein or peptide from the same protein, with the presence of an amino acid modification. The intensities plot of m/z 5442.5 was lower in the DHi, while those of m/z 8565.0 were higher in the DHo. Furthermore, the ionic map of these ions indicated that their intensities were not homogenous on the tissue section but showed spatial specificity, which could not be seen without imaging by mass spectrometry.

ATR-FTIR = Attenuated

Then, the predictive model, mainly based on these ion intensities, was used to classify the spectral data from NH. Thanks to this approach, the spectral data from NH could be classified as being closer to the DHi or the DHo. Spectral data using this predictive model were classified for every spectrum of the sections from NH and were represented as a color map: the spectrum belonging to the DHi in red and the spectra belonging to the DHo in yellow (Figure 1C).

Plasma protein fingerprint to predict the PSE-like muscle defect

The second part of the study aimed to assess the capacity of protein fingerprinting by MALDI-TOF mass spectrometry and ATR-FTIR spectroscopy to predict the PSE-like muscle defect in hams. Plasma samples were chosen since it is essential not to depreciate the carcass and to develop an easy-to-use method, blood sampling being easier than muscle biopsies. In the first part of this study, the prediction capacity of both spectral approaches was assessed separately using multivariate analysis. Then, both data matrixes were analyzed using the most discriminant spectra features to determine the synergy of both of these spectral methods. The predictive model built using neural network analysis from MALDI-TOF spectra showed a classification accuracy of 84.6% and a precision and recall of 88% and 84.6%, respectively, resulting in the correct classification of 100% of NH samples, whereas 2 DH samples were misclassified (Tables 12). This misclassification has to be moderated by the number of observations and by their corresponding score probability. Indeed, in both cases, the score probabilities were 56.4% and 65.5% for the DH class and 43.6% and 34.5% for the NH class. The neural network built with FTIR spectra showed a classification accuracy of 84.6% and a precision and recall of 88.5% and 84.6%, respectively. This resulted in the correct classification of 100% of DH samples, whereas 2 NH samples were identified as a false negative, classified as DH (Table 1), with a score probability of 66.1% and 96.9%.

To demonstrate the synergy of both spectral fingerprints in predicting the PSE-like muscle defect, both approaches were combined. The respective top 30 features found to be the most relevant in the neural networks obtained with MALDI-TOF and FTIR spectral fingerprints were analyzed to build a new predictive model. The results showed a classification accuracy of 100%, with a precision and recall of 100% and 100%. Thus, the classification results confirmed the relevance of such a combinatory approach since it improved the correct classification assignment up to 100%. All the individual spectra were correctly classified, meaning that the neural network successfully predicted the PSE-like muscle defect in all cases.

Conclusions

The spectral approaches used in this study have proven efficient to characterize the PSE-like muscle defect from the tissue to the molecular scales. This integrative study resulted in the definition of relevant protein markers to better understand the defect evolution within ham. Then, the spectral approaches were found to be highly discriminative in predicting PSE-like defect using a plasma sample and thus were without any depreciation of the carcass. Furthermore, this proof of concept has demonstrated the synergy of both spectral methods, thereby opening up new perspectives regarding their application in industrial conditions.

Figure 1 .

 1 Figure 1. Predictive model of the PSE-like areas. (A) Mean spectra acquired on the muscle sections from the "normal hams" (in blue, n = 10), on the muscle sections from the inner (in red, n = 10) and outer (in yellow, n = 10) parts of "PSE-like hams." The peaks with the highest loading scores and biggest contribution to the predictive model are represented by stars. (B) Box plot and graphical representation of all the individual peak intensities observed on the muscle sections, and ionic map for m/z 4128.7, m/z 4155.6, m/z 5442.5, and m/z 8565.0 for representative tissue section samples from "DHo," "DHi," and "NH." (C) NH spectral data classification using the predictive model: the spectra from NH classified as DHi are shown in red, and the spectra from NH classified as DHo are shown in yellow. Serial cross sections were stained with Sirius Red to reveal the connective network and with hematoxylin and eosin to reveal the tissue structure. DHi = inner parts of PSE-like hams; DHo = outer parts of PSE-like hams; NH = normal hams; PSE = Pale Soft Exudative.

  Total Reflectance-Fourier Transform InfraRed; IR = Infrared; MALDI = Matrix-Assisted Laser Dissociation Ionization; MS = Mass Spectrometry; PSE = Pale Soft Exudative; TOF = Time of Flight.Meat and Muscle Biology 2020, 4(2): 13, 1-6 Théron et al. Insight into the molecular mechanisms American Meat Science Association. the spectral classes, in percentage, and its use as a tool to determine the class in which the new spectral data belonged. The m/z intensities on which the model's performance rely are those showing the highest scores: m/z 4128.7, m/z 4155.6, m/z 5442.5, and m/z 8565.0. These 4 peaks are represented in Figure 1A with stars on the mean spectra. The two first peaks, m/z 4128.7 and m/z 4155.6, are significantly more intense in the DHi compared with the DHo and those of the NH.

Table 1 .

 1 Evaluation and results of the predictive model, using a neural network algorithm, to classify PSE-like muscle defect and normal hams

		MALDI-TOF MS spectral					MALDI-TOF MS and ATR-FTIR
		fingerprint		ATR-FTIR spectral fingerprint	spectral fingerprints
			Evaluation results of the predictive model by neural network algorithm
	AUC		1.000			1.000			1.000
	CA		0.846			0.846			1.000
	F-1		0.840			0.844			1.000
	Precision		0.880			0.885			1.000
	Recall		0.846			0.846			1.000
			Classification results using the predictive model by neural network algorithm
			Score				Score		Score
	Identification	Classification assignment	probability PSE N	Classification assignment	probability PSE N	Classification assignment	probability PSE N
	N17	N	0,218	0,782	PSE	0.661	0.339	N	0.011	0.989
	N30	N	0,238	0,762	N	0.090	0.910	N	0.033	0.967
	N41	N	0.064	0.936	N	0.013	0.987	N	0.001	0.999
	N42	N	0.013	0.987	N	0.006	0.994	N	0.002	0.998
	N54	N	0.015	0.985	PSE	0.969	0.031	N	0.095	0.905
	N78	N	0.254	0,746	N	0.005	0.995	N	0.009	0.991
	N91	N	0.385	0,615	N	0.014	0.986	N	0.013	0.987
	PSE24	PSE	0.996	0.004	PSE	0.949	0.051	PSE	0.957	0.043
	PSE47	PSE	0.973	0.027	PSE	0.988	0.012	PSE	0.993	0.007
	PSE50	PSE	0.996	0.004	PSE	0.995	0.005	PSE	0.999	0.001
	PSE68	N	0.345	0.655	PSE	0.994	0.006	PSE	0.999	0.001
	PSE77	N	0.436	0,564	PSE	0.761	0.0,239	PSE	0.516	0.484
	PSE80	PSE	0.999	0.001	PSE	0.629	0.371	PSE	0.999	0.001

Table 2 .

 2 Evaluation and results of the predictive model, using a neural network algorithm, to classify PSE-like muscle defect and normal ham, including all the m/z class attributes

	Class	N	N	N	N	N	N	N	D	D	D	D	D	D
	Identification	N17	N30	N41	N42	N54	N78	N91	PSE24	PSE47	PSE50	PSE68	PSE77	PSE80
	Neural Network	N	N	N	N	N	N	N	P S E	P S E	P S E	N	N	P S E
	Neural Network (D)	0.218	0.238	0.064	0.013	0.015	0.254	0.385	0.996	0.973	0.996	0.345	0.436	0.998
	Neural Network (N)	0.782	0.762	0.936	0.987	0.985	0.746	0.615	0.004	0.027	0.004	0.655	0.564	0.002
	m/z							Class Attributes
	1123.45	0.71	0.45	0.93	0.40	0.78	0.91	0.93	0.71	1.13	1.19	1.68	0.48	1.59
	1141.52	0.27	0.53	0.53	0.28	0.15	0.92	0.90	0.64	1.39	1.97	0.45	0.22	2.45
	1159.83	0.16	0.38	0.36	0.24	0.15	0.55	0.67	0.48	0.90	1.60	0.27	0.20	2.15
	1409.07	0.29	0.30	0.54	0.20	0.14	0.55	0.53	0.32	0.79	1.26	0.51	0.20	1.47
	1547.11	0.19	0.16	0.24	0.15	0.17	0.62	0.34	0.30	0.67	1.15	0.18	0.25	1.76
	1623.01	0.29	0.39	0.43	0.30	0.34	0.71	0.69	0.54	1.04	1.75	0.47	0.32	2.30
	1811.76	0.17	0.35	0.19	0.19	0.16	0.43	0.43	0.39	0.56	0.96	0.21	0.26	0.86
	1998.73	0.48	0.62	0.73	0.46	0.70	0.66	0.63	0.71	0.71	0.85	1.06	0.48	0.97
	2154.31	3.47	2.92	3.45	1.81	7.20	2.35	1.23	0.66	0.76	0.99	0.50	0.74	1.23
	2239.21	0.50	0.30	0.39	0.21	0.37	0.59	0.16	0.71	0.80	0.43	0.77	0.31	0.69
	2287.61	0.29	0.19	0.26	0.19	0.30	0.64	0.35	0.56	0.41	0.72	0.48	0.29	1.25
	2378.15	0.61	1.41	0.50	0.59	0.96	0.80	1.34	1.43	1.25	1.24	1.38	1.08	1.33
	2574.58	0.30	0.81	0.35	0.28	0.47	0.87	0.62	0.82	1.10	0.87	0.49	0.80	0.89
	2701.67	0.44	0.58	0.40	0.31	0.47	0.44	0.44	0.55	0.51	0.63	0.48	0.52	0.56
	3277.30	0.52	1.10	0.67	0.87	2.01	0.89	0.78	1.78	0.87	1.75	2.34	1.31	2.10
	3476.56	0.66	0.16	0.29	0.20	0.37	0.45	0.33	0.61	0.37	0.49	0.44	0.79	1.49
	4564.62	0.14	0.23	0.18	0.50	1.19	0.54	0.79	3.59	0.89	2.93	2.73	1.38	3.61
	5182.11	0.07	0.09	0.06	0.05	0.35	0.31	0.20	0.04	0.04	0.05	0.03	0.08	0.08
	5601.50	0.05	0.05	0.02	0.03	0.08	0.19	0.04	0.16	0.10	0.09	0.07	0.16	0.24
	6948.88	0.89	0.11	0.30	0.44	0.51	0.72	0.56	1.29	0.54	0.54	0.97	2.49	3.40
	PSE = Pale Soft Exudative.

American Meat Science Association.2 www.meatandmusclebiology.com

www.meatandmusclebiology.com

American Meat Science Association.


Yuandou Wang

Baogang Zhang

Xin ; Shen

Qian Li

Feng Su
email: sufeng@qust.edu.cn

Suming M Li
email: suming.li@umontpellier.fr

Biocompatibility, drug release and anti-tumor effect of pH-sensitive micelles prepared from poly(2-ethyl-2-oxazoline)-poly(DL-lactide) block copolymers

Keywords: poly(2-ethyl-2-oxazoline), poly(DL-lactide), self-assembly, micelle, curcumin poly(2-ethyl-2-oxazoline), poly(DL-lactide), self-assembly, micelles, curcumin, nano carrier

    Biocompatibility, drug release, and anti-tumor effect of pH -sensitive micelles prepared from poly(2-ethyl-2-oxazoline)-poly( DL -lactide) block copolymers

Introduction

Nowadays, cancer is one of the most common causes of death globally. Nearly 10 million of people died of cancer in 2020 according to the World Health Organization. [START_REF] Siegel | Cancer statistics[END_REF][START_REF] Sung | Global cancer statistics 2020: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries[END_REF][START_REF] Zheng | Report of cancer epidemiology in China, 2015[END_REF] .

For patients with different types of solid tumors, the most commonly used first-line clinical treatment is combination of surgery and chemotherapy. However, one of the main problems of chemotherapy is the extremely low bioavailability of anti-tumor drugs and adverse side effects. [START_REF] Aquib | Advances in local and systemic drug delivery systems for postsurgical cancer treatment[END_REF][START_REF] Christiana | Chemotherapy-induced cardiotoxicity: New insights into mechanisms, monitoring, and prevention[END_REF][START_REF] Hanovich | Rechallenge strategy in cancer therapy[END_REF] Many nano-platforms such as micelles, liposomes, vesicles and nanoparticles have been designed to prevent drug deterioration while increasing effective drug concentration at lesion site. [START_REF] Wan | Polymeric micelles with reduction-responsive function for targeted cancer chemotherapy[END_REF][START_REF] Jin | Microwave-triggered smart drug release from liposomes co-encapsulating doxorubicin and salt for local combined hyperthermia and chemotherapy of cancer[END_REF][START_REF] Wang | NIR-absorbing dye functionalized supramolecular vesicles for chemo-photothermal synergistic therapy[END_REF][START_REF] Lu | Smart cancer cell targeting imaging and drug delivery system by systematically engineering periodic mesoporous organosilica nanoparticles[END_REF] Among them, micelles have attracted much interest due to their advantageous characteristics, including good stability in vivo, long circulation in the bloodstream, sustained and adjustable drug release performance. A number of micelles prepared from amphiphilic block copolymers, have been brought to the stage of clinical trials. [START_REF] Hasenstein | Antitumor activity of Triolimus: a novel multidrug-loaded micelle containing Paclitaxel, Rapamycin, and 17-AAG[END_REF][START_REF] Fletcher | Next generation polymeric nanomedicines for oncology: Perspectives and future directions[END_REF][START_REF] Khanna | A review of paclitaxel and novel formulations including those suitable for use in dogs[END_REF] Drugs encapsulated in micelles can be delivered at the target site through drug diffusion and copolymer degradation [START_REF] Fan | Recent progress of crosslinking strategies for polymeric micelles with enhanced drug delivery in cancer therapy[END_REF][START_REF] Varela-Moreira | Clinical application of polymeric micelles for the treatment of cancer[END_REF] . Additionally, the chemical structure of micelle material can be designed to tailor the degradation rate and, thus allowing spatiotemporally controlled drug release.

Curcumin is a natural polyphenol extracted from the rhizome of turmeric (Curcuma longa) which exhibits various pharmacological activities because of its versatile molecular targets. [START_REF] Bandyopadhyay | Farmer to pharmacist: Curcumin as an anti-invasive and antimetastatic agent for the treatment of cancer[END_REF] Curcumin has been widely explored in preventing or treating different types of diseases, such as cancer, gastrointestinal diseases, as well as gastrointestinal and arthritis-related disorders. [START_REF] Mehta | Curcumin and lung cancer-a review target[END_REF][START_REF] Mirzaie | Anticancer drug delivery systems based on curcumin nanostructures: A review[END_REF][START_REF] Najafi | The role of curcumin/curcuminoids during gastric cancer chemotherapy: A systematic review of non-clinical study[END_REF][START_REF] Ashrafizadeh | Curcumin in cancer therapy: A novel adjunct for combination chemotherapy with paclitaxel and alleviation of its adverse effects[END_REF] However, the very low solubility (11 ng mL -1 ), fast metabolism and clearance of curcumin lead to low bioavailability, which considerably limits its clinical applications. It is thus of major importance to develop a nano carrier to encapsulate curcumin so as to improve its bioavailability.

Poly(2-ethyl-2-oxazoline) (PEOz) is a hydrophilic polymer with aliphatic side chains and a skeleton structure similar to natural polypeptides. It has been approved by the food and drug administration (FDA) as a food additive. [START_REF] Viegas | Polyoxazoline: Chemistry, properties, and applications in drug delivery[END_REF] Due to its biosafety and hydrophilicity, PEOz is considered as an excellent substitute to poly(ethylene glycol) (PEG) in the field of biomedical research. Micelles prepared from PEOz-based block copolymers present attractive properties, including long circulation time, sustained drug release and no accumulation in normal organs. [START_REF] Bauer | Poly(2-ethyl-2-oxazoline) as alternative for the stealth polymer poly(ethylene glycol): Comparison of in vitro cytotoxicity and hemocompatibility[END_REF][START_REF] Mero | Synthesis and characterization of poly(2-ethyl-2-oxazoline)-conjugates with proteins and drugs: Suitable alternatives to PEG-conjugates[END_REF] Meanwhile, ionization of tertiary amide groups leads to electrostatic repulsion between PEOz chains that provides PEOz-based micelles with pH sensitivity. Zhao et al. synthesized a PEOzpoly(DL-lactide) (PEOz-PDLLA) copolymer composed of a PEOz block with Mn of 6514 and a PDLLA block with Mn of 8200. [START_REF] Zao | pH-responsive polymeric micelles based on poly(2-ethyl-2-oxazoline)poly(D,L-lactide) for tumor-targeting and controlled delivery of doxorubicin and P-glycoprotein inhibitor[END_REF] Doxorubicin loaded micelles were prepared using dialysis method. Drug release studies were performed at pH 7.4, 6.5 and 5.0 to simulate the blood environment, the tumoral extracellular environment, and the endo/lysosome mimetic circumstance, respectively. All the designed micelles exhibited an accelerated drug release in acidic pH media as doxorubicin was almost totally released beyond 12 h at pH 5.0. [START_REF] Zao | pH-responsive polymeric micelles based on poly(2-ethyl-2-oxazoline)poly(D,L-lactide) for tumor-targeting and controlled delivery of doxorubicin and P-glycoprotein inhibitor[END_REF] In our previous work, a number of PEOz-poly(Llactide) (PEOz-PLLA) copolymers were synthesized as pH-sensitive carrier of paclitaxel. [START_REF] Su | Novel selfassembled micelles of amphiphilic poly (2-ethyl-2-oxazoline)-poly (L-lactide) diblock copolymers for sustained drug delivery[END_REF] It was observed that the hydrophilic/hydrophobic balance and hydrophobic block length of copolymers significantly affect the micelle formation and paclitaxel release behavior. Spherical micelles were exclusively observed for PEOz-PLLA copolymers with short PLA blocks, whereas both worm-like and spherical micelles were obtained for copolymers with long PLA blocks. But drug release was rather slow with less than 40% of drug released after 30 days.

In the present work, PEOz-PDLLA diblock copolymers with various compositions were prepared using monohydroxyl terminated PEOz as macro-initiator. Nano-sized micelles were obtained by self-assembly of the resulted copolymers in water using chloroform as a co-solvent, and characterized from dynamic light scattering (DLS), transmission electron microscopy (TEM), and critical micelle concentration (CMC) measurements. The cytocompatibility, heamocompatibility and Zebrafish embryo compatibility of the copolymers were studied in detail. Using curcumin as a drug model, the drug encapsulation properties and drug release behaviors of micelles were evaluated under in vitro conditions. Finally, the anti-tumor activity of curcumin loaded PEOz-PDLLA micelles on lung cancer cells was assessed for their potential applications in cancer therapy.

EXPERIMENTAL

Materials

DL-lactide was synthesized by polycondensation of DL-lactic acid, followed by thermal composition and cyclization according to literature method. [START_REF] Zhang | Preparation of monomer of degradable biomaterial poly(L-lactide)[END_REF] Monohydroxyl terminated poly(2-ethyl-2-oxazoline) was prepared by ring-opening polymerization of 2-ethyl-2-oxazoline. [START_REF] Lee | Phase-transition characteristics of amphiphilic poly(2-ethyl-2-oxazoline)/poly(ε-caprolactone) block copolymers in aqueous solutions[END_REF]

Synthesis of PEOz-PDLLA copolymers

Monohydroxyl terminated PEOz-OH was first synthesized according to our previously reported work. [START_REF] Su | Novel selfassembled micelles of amphiphilic poly (2-ethyl-2-oxazoline)-poly (L-lactide) diblock copolymers for sustained drug delivery[END_REF] The M n of obtained PEOz-OH was 2800 g/mol. PEOz-PDLLA block copolymers were synthesized in chlorobenzene according to the following procedure. Typically, PEOz-OH (1.0 g), DL-lactide (0.58 g), and stannous octoate (5 mg) were introduced in a flask, followed by addition of chlorobenzene (15 mL). Ring opening polymerization then occurred at 120 °C with inert gas protection.

After 30 h reaction, the PEOz-PDLLA copolymer was obtained by filtration through silica gel, precipitation in cold diethyl ether, and vacuum drying in oven at 40°C up to constant weight.

Self-assembly of PEOz-PDLLA copolymers

Co-solvent method was employed to prepare self-assembled PEOz-PDLLA micelles. Typically, PEOz-PDLLA copolymer (20 mg) was introduced in chloroform (1 mL), and solubilized. The resulted copolymer solution was then dropped in ultrapure water (20 mL). Vigorous stirring was applied for 4 h so as to allow self-assembly of copolymer micelles and evaporation of the solvent. A micelles solution at 1.0 mg/mL was finally obtained by filtration via a 0.22 µm filter membrane.

Characterization

Proton nuclear magnetic resonance ( 1 H NMR) was performed using a Bruker Avance III instrument. 5 mg of sample were dissolved in 0.6 mL deuterated chloroform (CDCl 3 ). And the spectra were recorded at 500 MHz with tetramethylsilane as an internal reference.

Gel permeation chromatography (GPC) was carried out on a Shimadzu apparatus equipped with a Waters 410 refractometer. 10 mg of sample were dissolved in 1 mL tetrahydrofuran taken as mobile phase. Measurements were made at 25°C, and calculations were realized based on a calibration curve previously obtained using Transmission electron microscopy (TEM) was performed on JEM 1200EX microscope (JEOL, Japan). A micelle solution (5 µL) at 1.0 mg/mL was dropped onto a carbon coated copper grid, followed by negative staining with a phosphotungstic acid solution at 1.0 wt.%. The sample was air dried, and then examined by TEM at an acceleration voltage of 80 kV.

Dynamic light scattering (DLS) was carried out by using a Nano-ZS90 nanosizer (Malvern, UK). Micelle solutions at 1.0 mg/mL were filtered via a 0.45 μm PTFE microfilter before measurements made at room temperature. The scattering angle was fixed at 90°. The critical micelle concentration (CMC) of PEOz-PDLLA copolymers was determined using fluorescence spectrophotometer (F-4600). Pyrene was taken as a fluorescent probe. First, 1 mL pyrene solution in benzene (2 × 10 -5 M) was poured into a 10 mL flask, followed by solvent evaporation. Various volumes of micelle solution and ultrapure water were added to the flask to a total volume of 10 mL. The final concentration of micelles was in the range from 1.0×10 -3 to 0.5 mg/mL, and that of pyrene was 2.0×10 -6 M. After 24 h equilibrium at 25°C, the excitation spectra of the solutions were registered in the range from 350 to 450 nm at a wavelength of 334 nm.

The intensity ratio at 375 and 395 nm was plotted vs copolymer concentration, and the CMC value was derived from the cross-over point of the two regression lines.

Biocompatibility studies

Hemolysis test

A micelle solution (10 mL) at 1.0 mg/mL was poured in a beaker which was previously silanized. The solution was heated at 37 ℃ for a period of 30 min. 0.2 mL rabbit blood diluted with acid-citrate-dextrose (ACD) at a dilution ratio of 9/1 was then added. The mixture was incubated at 37 ℃ for 30 min, and then centrifuged. The optical density (OD) of the supernatant was measured at 540 nm. Physiological saline was taken as negative control, and ultrapure water as positive control. The hemolysis The dynamic clotting time was determined according to the method previously reported in literature. [START_REF] Wang | Bioresorbable hydrogels prepared by photo-initiated crosslinking of diacrylated PTMC-PEG-PTMC triblock copolymers as potential carrier of antitumor drugs[END_REF] A micelle solution (25 µL) in 0.025 M calcium chloride (CaCl 2 ) was poured into a siliconized tube. After 5 min incubation at 37℃, ACD solution (200 µL) was introduced into the tube and mixed. At preset time points up to 140 min, distilled water (20 mL) was gently poured into the tube to rinse the watch-glass.

Subsequently, the absorbance of the rinsing liquid was measured at 490 nm wavelength.

Similar analyses were made on siliconized glass as negative control and ordinary glass as positive control. The clotting time of micelle samples was obtained from the OD value vs time plots. Experiments were repeated three times in each case.

MTT assay

MTT assay was employed to examine the cytotoxicity of copolymers, using L929 fibroblast cell line. Various micelle solutions at a concentration from 0.05 to 1.0 mg/mL with 10% calf serum were exposed to L929 cells in 96-well plates. At preset time intervals (24, 48 and 72 h), MTT (20 μL) at 5 mg/mL was added in the well, and incubated for 6 h. The medium was then taken out, followed by addition of 150 μL dimethyl sulfoxide (DMSO). The plates were shaken during 10 min. And the OD value was measured at 570 nm in a microplate reader. A phenol solution (100 μL) was taken as positive control, and a fresh medium (100 μL) as negative control. The relative growth rate (RGR) of cells was obtained according to the following equation:

(2) 𝑅𝐺𝑅(%) = 𝑂𝐷 𝑡𝑒𝑠𝑡 𝑠𝑎𝑚𝑝𝑙𝑒 𝑂𝐷 𝑛𝑒𝑔𝑎𝑡𝑖𝑣𝑒 𝑐𝑜𝑛𝑡𝑟𝑜𝑙 × 100%

Drug release studies

Curcumin was loaded in the core of micelles using a two-step procedure. Drug free micelles were first prepared as described in Section 2.3. 1 mg curcumin was dissolved in 1 mL methanol, and the solution was then introduced into 10 mL micelle Curcumin release from PEOz-PDLLA micelles was studied under in vitro conditions according to literature method. [START_REF] Lu | Selfassembled micelles prepared from bio-based hydroxypropyl methyl cellulose and polylactide amphiphilic block copolymers for anti-tumor drug release[END_REF][START_REF] Lu | Synthesis and self-assembly of hydroxypropyl methyl cellulose-block-poly(ε-caprolactone) copolymers as nanocarriers of lipophilic drugs[END_REF] A dialysis system was employed, which includes Float-A-Lyzer G2 dialysis devices (MWCO of 3500 Da), an oscillator and a thermostat. 1 mL curcumin loaded micellar solution was introduced in a dialysis device which was immersed in a centrifugation tube filled with 40 mL phosphate buffered saline (PBS). The tubes were fixed in an oscillator thermostated at 37℃. Drug release then proceeded under constant shaking. Sink conditions were ensured by regular renewal of the release medium. At each time point, a volume of 25 μL was taken out and equal volume of PBS was added. The collected samples were lyophilized, and dissolved in ethanol. The UV absorbance was measured to determine the drug concentration based on a previously established calibration curve. The drug loading content (LC) and loading efficiency (LE) were calculated from the following equations:

(3) 𝐿𝐶(%) = 𝑤𝑒𝑖𝑔ℎ𝑡 𝑜𝑓 𝑙𝑜𝑎𝑑𝑒𝑑 𝑑𝑟𝑢𝑔 𝑤𝑒𝑖𝑔ℎ𝑡 𝑜𝑓 𝑑𝑟𝑢𝑔 𝑙𝑜𝑎𝑑𝑒𝑑 𝑚𝑖𝑐𝑒𝑙𝑙𝑒𝑠 × 100% (4) 𝐿𝐸(%) = 𝑤𝑒𝑖𝑔ℎ𝑡 𝑜𝑓 𝑙𝑜𝑎𝑑𝑒𝑑 𝑑𝑟𝑢𝑔 𝑡ℎ𝑒𝑜𝑟𝑒𝑡𝑖𝑐𝑎𝑙 𝑑𝑟𝑢𝑔 𝑙𝑜𝑎𝑑𝑖𝑛𝑔 × 100%

In vitro antitumor activity

The anti-tumor activity of free curcumin and curcumin loaded micelles was determined by MTT assay using A549 lung cancer cell line. A549 cells were suspended in Dulbecco's Modified Eagle Medium (DMEM) which contains 10% fetal bovine serum (FBS), 100 U/mL penicillin and 100 µg/mL streptomycin, and incubated at 37 °C during 24 h in a humidified environment with 5% carbon dioxide. MTT assay was then realized according to the experimental protocol described above.

M n = M nPEOz +DP PDLLA ×72 (7) where 99 designates the molar mass of 2-ethyl-2-oxazoline moieties, and 72 that of lactyl moieties. and EOz 28 DL 38 , respectively. The M nNMR derived from NMR spectra varies from 3700 to 5500 g/mol. GPC was also used to characterize the copolymers as it allows to determine not only the molar mass, but also the molar mass distribution. As shown in Fig. 2, the GPC chromatograms shift to shorter retention time with increasing PDLLA block length, in agreement with higher molar mass. M nGPC values are slightly different from M nNMR ones because the former are derived from the hydrodynamic volume of polymers in solution compared to standards (Table 1). The dispersity (Ð) is in the range from 1.15 to 1.39, indicating that the copolymers have a narrow distribution of molar masses. PEOz corona and a hydrophobic PDLLA core. TEM was used to observe the morphology of self-assembled aggregates as shown in Fig. 3A-C. All copolymer micelles exhibit a spherical shape and are uniformly distributed. The diameter of micelles is in the range from 20 to 30 nm as estimated from TEM images. No wormlike micelles were observed, in contrast to PEOz-PLLA copolymers. [START_REF] Su | Novel selfassembled micelles of amphiphilic poly (2-ethyl-2-oxazoline)-poly (L-lactide) diblock copolymers for sustained drug delivery[END_REF] In fact, polymer chain structure regularity strongly affects the self-assembled morphology of micelles.

PDLLA blocks have a disordered chain structure with randomly distributed L-and Dlactidyl units, and PEOz-PDLLA copolymers seem to be more inclined to form spherical micelles as compared to PEOz-PLLA copolymers with rigid stereoregular PLLA blocks. The average micelle diameter and polydispersity index (PDI) were determined DLS measurements (Fig. 3). A symmetric size distribution is detected for all copolymer micelles, as shown in Fig. 3D-F. PDLLA copolymer with Mn of 6514 for PEOz and 8200 for PDLLA, [START_REF] Zao | pH-responsive polymeric micelles based on poly(2-ethyl-2-oxazoline)poly(D,L-lactide) for tumor-targeting and controlled delivery of doxorubicin and P-glycoprotein inhibitor[END_REF] which could be assigned to the more compact micelle core due to longer PDLLA block length.

The CMC is one of the key parameters of micelles as it allows to assess their stability in vivo after dilution caused by intravenous injection. As shown in

In vitro drug release

Due to its excellent anti-tumor activity and unique physico-chemical properties, curcumin was selected as a model of hydrophobic anti-tumor drugs. The nano-drug delivery system containing curcumin was prepared in two steps. [START_REF] Mansouri | Clinical effects of curcumin in enhancing cancer therapy: A systematic review[END_REF] Blank micelles were first obtained by using co-solvent evaporation method, and a curcumin solution in methanol was then added into the micelle solution. Drug loaded nano-micelles were obtained under vigorous stirring to remove the solvent.

The LC and LE data of curcumin loaded micelles are summarized in Table 2 endo/lysosome mimetic environment. As shown in Fig. 6, two distinct release phases were observed in the release curves of drug loaded PEOz-PDLLA micelles. An initial fast release of curcumin was detected in the first 12 h, which could be attributed to the fact that drug molecules are partly located at the interface of the core-corona structure. [START_REF] Tyrrell | Fabrication of micellar nanoparticles for drug delivery through the self-assembly of block copolymers[END_REF] Subsequently, a stable and sustained release behavior was detected up to 144 h. At pH and found that the degradation of micelles strongly affects drug release rate. [START_REF] Jelonek | Effect of polymer degradation[END_REF] In our previous study on PLGA-PEG copolymer micelles, faster degradation was observed for micelles with shorter PLGA blocks as compared to micelles with longer PLGA blocks. [START_REF] Su | Effects of chemical composition on the in vitro degradation of micelles prepared from poly(D,Llactide-co-glycolide)-poly(ethylene glycol) block copolymers[END_REF] Thus, the various drug release rates obtained for EOz 28 DL 13 , EOz 28 DL 29 and different pH values. [START_REF] Zao | pH-responsive polymeric micelles based on poly(2-ethyl-2-oxazoline)poly(D,L-lactide) for tumor-targeting and controlled delivery of doxorubicin and P-glycoprotein inhibitor[END_REF] The acidic environment promoted the release of doxorubicin because of higher water-solubility of doxorubicin and accelerated degradation of the hydrophobic PDLLA block. Almost total drug release was achieved within 24 h. In our previous work, a series of PEOz-PLLA micelles were evaluated for controlled release of paclitaxel which is one of the most widely studied and most efficace anti-tumor drugs.

Within a 30-day period, copolymers with shorter PLLA blocks exhibit faster drug release than those with longer PLLA blocks. Faster drug release is also obtained at acidic pH than at pH 7.4. Nevertheless, the highest release ratio was only 37.6% which was obtained for EOz 18 L 5 micelles after 30 days release in PBS at pH 5.0. Therefore, PEOz-PDLLA micelles present much faster drug release than PEOz-PLLA ones, which in consistent with the faster degradation rate of PDLLA compared to PLLA. [START_REF] Su | Novel selfassembled micelles of amphiphilic poly (2-ethyl-2-oxazoline)-poly (L-lactide) diblock copolymers for sustained drug delivery[END_REF]

In vitro antitumor studies

Lung cancer is one of the most deadly malignant tumors for both women and men, making up nearly 25% of all cancer deaths. The inhibitory effect of curcumin loaded micelles on the proliferation of A549 lung cancer cells is studied to evaluate their potential for lung tumor treatment. As shown in Fig. 7A, no cytotoxicity was detected for PEOz-PDLLA micelles (0.25-250 μg) after 72 h co-culture with A549 cells. The relative proliferation rate of A549 cells in micelle solutions was similar to that in fresh medium. This result is consistent with the good cytocompatibility of PEOz-PDLLA micelles described above.

Fig. 7B presents the inhibitory effect of free curcumin and curcumin loaded micelles on the proliferation of A549 cells. The concentration of free drug is the same as that in the micellar system, taking into account the drug loading content of micelles.

For low concentrations of curcumin from 0.025 to 2.5 µg/mL, cell proliferation was significantly inhibited and showed a dose-dependent decrease with increasing concentration. In contrast, little RGR changes were detected in the drug concentration range of 2.5-25 μg/ml. In the whole drug concentration range, the anti-tumor effect of curcumin loaded micelles was comparable to that of free curcumin.

  ) was obtained using the following equation: (1) 𝐻𝑅(%) = 𝑂𝐷 𝑡𝑒𝑠𝑡 𝑠𝑎𝑚𝑝𝑙𝑒 -𝑂𝐷 𝑛𝑒𝑔𝑎𝑡𝑖𝑣𝑒 𝑐𝑜𝑛𝑡𝑟𝑜𝑙 𝑂𝐷 𝑝𝑜𝑠𝑖𝑡𝑖𝑣𝑒 𝑐𝑜𝑛𝑡𝑟𝑜𝑙 -𝑂𝐷 𝑛𝑒𝑔𝑎𝑡𝑖𝑣𝑒 𝑐𝑜𝑛𝑡𝑟𝑜𝑙 × 100% 2.5.2 Dynamic clotting time

  stirring was applied during 4 h to ensure efficient drug loading, followed by gentle stirring for 24 h to completely eliminate the solvent. The resulting curcumin-loaded micelle solution was centrifuged to remove unloaded drug, and filtered through 0.22 μm filter.

1

 1 Characterization of PEOz-PDLLA copolymers PEOz-PDLLA copolymers were prepared by ring-opening polymerization of DLlactide using monohydroxy terminated PEOz-OH with M n of 2800 as macro-initiator and Sn(Oct) 2 as catalyst. The chemical composition of PEOz-PDLLA copolymers was determined by 1 H NMR, as shown in Fig. 1. Three main signals are detected for the PEOz block. Peak a at 1.12 ppm belongs to the methyl protons of ethyl side chain, peak b at 2.40 and 2.31 ppm to the methylene protons of ethyl side chain, and peak c at 3.46 ppm to the methylene protons of PEOz main chain, respectively. In the case of the PDLLA hydrophobic block, two signals are detected. Peak d at 5.17 ppm is attributed to the methine proton, and peak e at 1.59 ppm to the methyl protons of lactyl repeat units. The signals d and a are integrated using MestReNova software, and the integrations are used to calculate the LA/EOz molar ratio of PEOz-PDLLA copolymers. The degree of polymerization (DP) of both blocks and M n of PEOz-PDLLA are then obtained according to the following equations : DP PEOz = M nPEOz /99 (5) DP PDLLA = DP PEOz ×(LA/EOz)

Figure 1 . 1 H

 11 Figure 1. 1 H NMR spectrum of PEOz-PDLLA copolymer in CDCl 3

Figure 2 .

 2 Figure 2. The curves of GPC of PEOz-PDLLA copolymers

Figure 3 .

 3 Figure 3. TEM images and DLS graphs of EOz 28 DL 13 (A, D), EOz 28 DL 29 (B, E), and EOz 28 DL 38 (C, F) copolymer micelles.

3. 4 . 2

 42 Dynamic clotting time Fresh blood in vitro will initiate endogenous coagulation due to foreign bodies activating endogenous coagulation factors. The dynamic coagulation behavior of PEOz-PDLLA micelles and controls was evaluated from time dependent changes of absorbance, as shown in Fig. 4. 0.1 and 0.01 was defined as the initial clotting time (ICT) and the complete clotting time (CCT), respectively. The blood in contact with positive control was the first to undergo endogenous coagulation. The ICT and CCT of positive control were 23 min and 60 min, respectively. EOz 28 DL 13 , EOz 28 DL 29 and EOz 28 DL 38 micelles showed the same trend as the negative control. The OD values gradually decreased over time. The ICT of all PEOz-PDLLA micelles was about 80 min and that of the negative control was 90 min. Furthermore, the CCT of PEOz-PDLLA micelles and the negative control were almost the same (above 140 min), thus suggesting that PEOz-PDLLA micelles present outstanding anticoagulant properties.

Figure 4 .

 4 Figure 4. OD value changes of PEOz-PDLLA micelles as a function of time in comparison with the positive and negative controls.

3. 4 . 3

 43 MTT assayWithin a certain cell concentration range, the amount of formazan crystals formed by MTT is directly proportional to cell viability. Fig.5presents L929 cell viability data after 24, 48 and 72 h co-culture with PEOz-PDLLA micelles at various concentrations from 0.5 to 5.0 mg/mL. The positive control showed very low RGR values which decreased from 24% at 24 h to 12.4% at 72 h. However, the RGR values are over 95% for all micelles at different concentrations throughout the 72 h period, thus showing a cytocompatibility very close to the negative control (100%). Therefore, both hemocompatibility and cytocompatibility data showed that PEOz-PDLLA micelles are biocompatible for potential applications in drug delivery according to the ISO 10993.

Figure 5 .

 5 Figure 5. RGR of L929 cells after 24 (A), 48 (B) and 72 h (C) co-culture with PEOz-PDLLA micelles.

  Dialysis method was used to determine the drug release behavior of curcuminloaded micelles at different pH values. Three representative pH values were selected to simulate the environments of micelles entering the body: pH 7.4 when the micelles were intravenously injected into the blood environment, pH 6.5 when the micelles were endocytosed by tumor cells, and pH 5.0 when the micelles were transferred into the

  EOz 28 DL 38 micelles can be directly related to the degradation rate of copolymers. The lower the pH, the shorter the PDLLA block length, the faster the degradation of micelles and the faster the release of curcumin.

Figure 6 .

 6 Figure 6. In vitro release profiles of curcumin from EOz 28 DL 13 (A), EOz 28 DL 29 (B), EOz 28 DL 38 (C) copolymer micelles in PBS at pH = 7.4, 6.5 and 5.0 at 37 °C; and comparison of the release curves of the three copolymers at pH 5.0 (D). All data are represented as the mean ± SD (n = 3).

Figure 7 .

 7 Figure 7. RGR data of A549 cells after 72 h culture with blank micelles (A), free curcumin and curcumin loaded micelles (B) in comparison with the control (DMEM).

F

Table 1

 1 presents the characterization results of the three copolymers. The copolymers are abbreviated as EOz x DL y for the sake of simplicity, with the subscripts x and y representing the DP of PEOz and PDLLA blocks, respectively. The DP PEOz is 28 for the three copolymers, and DP PDLLA is 13, 29 and 38 for EOz 28 DL 13 , EOz 28 DL 29

Table 1

 1 Structural characterization of PEOz-PDLLA diblock copolymers

	Copolymer M nPEOz	LA/EOz	DP PEOz	DP PDLLA	M nNMR	M nGPC	Ð	HLB
	EOz 28 DL 13 2800	0.45 (0.6) a)	28	13	3700	3500	1.15 15.1
	EOz 28 DL 29 2800	1.05 (1.2)	28	29	4900	5300	1.34 11.4
	EOz 28 DL 38 2800	1.35 (1.6)	28	38	5500	5800	1.39 10.2
	a) Values in parentheses designate the LA/EOz molar ratios in the feed.

3.2 Self-assembly of PEOz-PDLLA diblock copolymers Amphiphilic PEOz-PDLLA diblock copolymers can spontaneously self-assemble in aqueous medium, leading to formation of nano-sized micelles with a hydrophilic

Table 2

 2

summarizes the micelle diameter and PDI data of the three copolymers. The diameter of micelles slightly increases with the increase of the PDLLA block length, from 104 nm for EOz 28 DL 13 to 131 and 136 nm for EOz 28 DL 29 and EOz 28 DL 38 , respectively. This finding could be attributed to the increasing hydrophobic block length which could attract more copolymer chains in micelles, as reported in literature.

[START_REF] Su | Novel selfassembled micelles of amphiphilic poly (2-ethyl-2-oxazoline)-poly (L-lactide) diblock copolymers for sustained drug delivery[END_REF]

A slight increase of micelle size is observed after encapsulation of curcumin. The micelle diameter slightly increases to 131, 135 and 145 nm for EOz 28 DL 13 , EOz 28 DL 29 and EOz 28 DL 38 , respectively. The PDI values of micelle sizes are below 0.30 in all cases, in agreement with narrow size distribution. It is noteworthy that the diameter values derived from DLS are much larger than those from TEM due to the dehydration or shrinkage of micelles during air drying before TEM observation. Zhao et al. obtained a smaller micelle diameter of 41-43 nm for PEOz-

Table 2

 2

	that copolymers with longer hydrophobic segments self-assemble more easily in
	aqueous medium, leading to more stable micelle structures.
	F
	o r
	P
	e e r
	R e v
	i e w

, the CMC value obtained from fluorescence spectroscopy is highly dependent on the PDLLA block length or the HLB of copolymers. The longer the PDLLA block, the lower the HLB, and the smaller the CMC value. This finding is attributed to the fact

Table 2 .

 2 Self-assembly and drug loading properties of PEOz-PDLLA micellesThe hemolytic ratio (HR) reflects the dissociation level of hemoglobin after in vitro coculture of materials with RBC. In Table3are summarized the OD and HR values of PEOz-PDLLA micelles, together with the OD values of both positive and negative controls. The HR value of EOz 28 DL 13 , EOz 28 DL 29 and EOz 28 DL 38 micelles is 3.4%, 3.8%

	Sample	Diameter	PDI	CMC (mg/mL) LC (%)	LE (%)
		(nm)
	EOz 28 DL 13	104	0.29	0.0065	-	-
	EOz 28 DL 29	131	0.26	0.0043	-	-
	EOz 28 DL 38	136	0.22	0.0039	-	-
	EOz 28 DL 13 -Cur	131	0.22	-	7.0	75.0
	EOz 28 DL 29 -Cur	135	0.18	-	7.3	79.3
	EOz 28 DL 38 -Cur	145	0.13	-	7.6	82.5
	3.4 Biocompatibility studies
	3.4.1 Hemolysis test
	As potential drug carrier for intravenous application, hemolysis test is of major
	importance as it indicates the interaction of biomaterials and red blood cells (RBC).
	and 3.5%, respectively. Therefore, the hemolytic properties of PEOz-PDLLA
	copolymers are acceptable for medical and pharmaceutical uses as the HR value is
	lower than 5%, the threshold defined by ISO 10993.
	Table 3 Hemolytic properties of PEOz-PDLLA micelles

.

  EOz 28 DL 13 presents an LE of 75.0% and an LC of 7.0%. Higher LE and LC values are obtained for EOz 28 DL 29 and EOz 28 DL 38 . EOz 28 DL 38 presents the highest LE and LC values of 82.5% and 7.6%, respectively. The increase of LE and LC with increasing PDLLA block length could be attributed to the larger core volume of micelles with longer PDLLA blocks.

  7.4, the drug release in first 12 h was about 36%, 31%, and 31% for EOz 28 DL 13 , EOz 28 DL 29 and EOz 28 DL 38 , respectively. At pH 5.0, the initial drug release at 12 h increased to 56%, 53% and 45% for EOz 28 DL 13 , EOz 28 DL 29 and EOz 28 DL 38, respectively. Intermediate rates were obtained at pH 6.5. Beyond 12 h, the three drug loaded micelle systems showed slower release rates. At 144 h, the final cumulative release of curcumin at pH 7.4, 6.5 and 5.0 was 59%, 81% and 93% for EOz 28 DL 13 , 52%, 72% and 85% for EOz 28 DL 29, and 48%, 61% and 73% for EOz 28 DL 38 , respectively.The drug release behaviors of three micelle systems at the same pH (pH 5) are comparatively shown in Fig.6D. In the first 12 h, the cumulative release curves of EOz 28 DL 13 and EOz 28 DL 29 almost overlap, while EOz 28 DL 38 shows lower release rate.

	In the later stage, drug release continues to increase in all three systems, but at a slower
	rate. At pH 5.0, the cumulative drug release was 93%, 85% and 73% for EOz 28 DL 13 ,
	EOz 28 DL 29 and EOz 28 DL 38 , respectively. Therefore, curcumin release from PEOz-
	PDLLA micelles is strongly affected by the environmental conditions and the
	composition or PDLLA block length of copolymers.
	The release data of curcumin from PEOz-PDLLA micelles clearly illustrate the
	responsiveness of micelles to pH stimulation. Drug diffusion and carrier degradation
	are the two main mechanisms that affect drug release behaviors. Jelonek et al. studied
	paclitaxel release from PLLA-PEG and PDLLA-PEG micelles at different pH values,

  on prolonged release of paclitaxel from filomicelles of polylactide/poly(ethylene glycol) block copolymers. Mater Sci Eng C. 2017; 75: 918-925.

	F F F F F
	o r o r o r o r o r o r
	P P P P P P
	e e r e e r e e r e e r e e r e e r
	R e v R e v R e v R e v R e v R e v
	i e w i e w i e w i e w i e w i e w

Acknowledgments

The work was financially supported by the Talent Fund of Shandong Collaborative Innovation Center of Eco-Chemical Engineering (XTCXQN20), the Shandong Provincial Natural Science Foundation (ZR2020QH271, ZR2020MH324), and the Postdoctoral Applied Research Project of Qingdao Municipality (04030431060088).


Qingzhen Zhu

Baogang Zhang

Yuandou Wang

Xinghua Liu

Weiwei Li

Feng Su
email: sufeng@qust.edu.cn

S M Li

Suming ; Li
email: suming.li@umontpellier.fr

Suming Li

Self-assembled micelles prepared from poly(D,L-lactide-co-glycolide)poly(ethylene glycol) block copolymers for sustained release of valsartan

Keywords: drug delivery systems, micelles, self-assembly, Poly(D,L-lactide-coglycolide), Poly(ethylene glycol) Poly(D,L-lactide-co-glycolide), Poly(ethylene glycol), Self-assembly, Micelles, Drug delivery

Self-assembled micelles prepared from poly(D,L-lactide-co-glycolide)-poly(ethylene glycol) block copolymers for sustained release of valsartan

valsartan, is a competitive antagonist of the angiotensin II (Ang II) receptor and member of angiotensin II (Ang II) receptor blockers (ARBs). [START_REF] Criscione | Pharmacological profile of valsartan: a potent, orally active, nonpeptide antagonist of the angiotensin II AT1-receptor subtype[END_REF] It is widely used for the treatment of essential and secondary hypertension and heart failure. Recently, it has been suggested that the inhibition of Ang II acting on angiotensin Ⅰ receptor (AT 1 R) may be beneficial to COVID-19 patients. [START_REF] Tignanelli | Antihypertensive drugs and risk of COVID-19?[END_REF] In fact, SARS-CoV-2 virus is susceptible to use angiotensin-converting enzyme 2 (ACE2) as a binding receptor which is widespread in human body. ACE2 cleaves Ang II and generates angiotensin 1-7 which is an efficient vasodilator. Ang II can bind to and then activate AT 1 R, while further promoting vasoconstriction. Sartan drugs will block excessive angiotensin-mediated AT1R activation due to the viral infection, so it will protect lung in anti-oxidant, antiinflammation, vasodilatation, and anti-fibrosis with increasing angiotensin 1-7.

Valsartan is a tetrazole derivative which contains two acidic groups with pKa of 4.73 and 3.9, respectively. [START_REF] Thomas | Design and characterization of valsartan co-crystals to improve its aqueous solubility and dissolution behavior[END_REF] Thus valsartan is soluble at physiological pH values in the form of undissociated acid, mono-anion, and di-anion. In fact, its water solubility is 0.18 g/L at 25˚C. In phosphate buffer at pH 8.0, valsartan has a solubility up to16.8 g/L at 25˚C. [START_REF] Saydam | Bioavailability file: valsartan[END_REF] Nevertheless, being acidic in nature, it is hardly soluble in the acidic environment of the gastrointestinal tract where is absorbed from the upper part, [START_REF] Siddiqui | Pharmacological and pharmaceutical profile of valsartan: a review[END_REF] causing lowered bioavailability. Various formulation strategies have been reported to improve the solubility of valsartan in order to achieve more beneficial pharmacokinetics, including solid dispersions, [START_REF] Xu | Enhanced dissolution and oral bioavailability of valsartan solid dispersions prepared by a freeze-drying technique using hydrophilic polymers[END_REF] cyclodextrin complexes, 7 self-microemulsifying system, [START_REF] Yeom | Enhanced oral bioavailability of valsartan using a polymer-based supersaturable self-microemulsifying drug delivery system[END_REF] nanostructured lipid carriers (NLC), [START_REF] Albekery | Optimization of a nanostructured lipid carriers system for enhancing the biopharmaceutical properties of valsartan[END_REF] hydrogels, [START_REF] Sohail | Development and in vitro evaluation of high molecular weight chitosan based polymeric composites for controlled delivery of valsartan[END_REF] transdermal gel containing vesicles, [START_REF] Ahad | The ameliorated longevity and pharmacokinetics of valsartan released from a gel system of ultradeformable vesicles[END_REF] and mesoporous silica nanoparticles. [START_REF] Biswas | Modified mesoporous silica nanoparticles for enhancing oral bioavailability and antihypertensive activity of poorly water soluble valsartan[END_REF] Drug loading of chitosan based hydrogels was realized by soaking dried gel in a drug solution, but the drug load content was rather low and drug release was very fast. [START_REF] Sohail | Development and in vitro evaluation of high molecular weight chitosan based polymeric composites for controlled delivery of valsartan[END_REF] Transdermal gel containing drug loaded vesicles were synthesized from Phospholipon® 90 G, Carbopol® 940, PEG-400 and triethanolamine. [START_REF] Ahad | The ameliorated longevity and pharmacokinetics of valsartan released from a gel system of ultradeformable vesicles[END_REF] The system could provoke skin irritation. NLC system successfully enhanced valsartan release rate and dissolution, but the system was not stable and the drug content was relatively low. [START_REF] Albekery | Optimization of a nanostructured lipid carriers system for enhancing the biopharmaceutical properties of valsartan[END_REF] In the case of formulations for oral delivery, sustained release of valsartan cannot be achieved despite enhanced oral bioavailability.

Therefore, it would be of great interest to develop drug delivery systems based on bioresorbable polymers as they allow to improve the pharmacokinetic and pharmacodynamic profiles of drugs, to minimize drug loss and/or degradation, and to prevent harmful side effects. Amphiphilic block copolymers composed of hydrophilic and hydrophobic blocks, including polylactide-poly(ethylene glycol) (PLA-PEG) and poly(lactide-coglycolide)-poly(ethylene glycol) (PLGA-PEG) are able to self-assemble to form micelles in an aqueous medium. The resulting nano-micelles present a core-corona structure with a hydrophobic core composed of PLA or PLGA segments which serve as a vessel for the incorporation of lipophilic drugs, [START_REF] Huh | PLGA-PEG block copolymers for drug formulations[END_REF][START_REF] Letchford | A review of the formation and classification of amphiphilic block copolymer nanoparticulate structures: micelles, nanospheres, nanocapsules and polymersomes[END_REF] while the hydrophilic PEG segments provide a protective corona which ensures the stability of micelles and prevents mononuclear phagocyte system devour. [START_REF] Letchford | A review of the formation and classification of amphiphilic block copolymer nanoparticulate structures: micelles, nanospheres, nanocapsules and polymersomes[END_REF] Some polymeric micelles have been used clinically. As a polymeric micelle formulation, Genexol ® -PM based on PLA-PEG copolymers with incorporated paclitaxel is under phase II clinical trials to evaluate its efficacy and safety in the treatment of recurrent or metastatic breast cancer. 16 Phase II clinical trials are also undertaken to compare the antitumor activity of Genexol ® -PM and Genexol ® combining cisplatin in non-small cell lung cancer. 17 Various aggregates can be obtained from self-assembly of amphiphilic block copolymers, including spherical micelles, rod-like micelles or cylinders, worm-like micelles or filo micelles, and polymersomes. [START_REF] Cameron | EWR Steacie Award Lecture Asymmetric amphiphilic block copolymers in solution: a morphological wonderland[END_REF] The architecture of self-assembled aggregates is mainly determined by the hydrophilic/hydrophobic ratio of block copolymers. In the case of PLA-PEG copolymers, spherical and worm-like micelles are obtained for copolymers with high or low PEG fraction, respectively. Importantly, the morphology of these nano-sized aggregates has a substantial influence on their performance in pharmacokinetics and biodistribution. [START_REF] Allen | Nano-engineering block copolymer aggregates for drug delivery[END_REF][START_REF] Croy | Polymeric micelles for drug delivery[END_REF] Worm-like micelles show surprisingly persistent circulation up to one week in the bloodstream, which is much longer compared to other nano-carriers, including stealthy vesicles bearing similar PEG chains. [START_REF] Geng | Shape effects of filaments versus spherical particles in flow and drug delivery[END_REF] PLGA-PEG copolymers have been largely investigated as a bioresorbable drug carrier because of their excellent degradability. [START_REF] Jeong | Biodegradable thermosensitive micelles of PEG-PLGA-PEG triblock copolymers[END_REF][START_REF] Chen | PLGA-PEG-PLGA triblock copolymeric micelles as oral drug delivery system: In vitro drug release and in vivo pharmacokinetics assessment[END_REF][START_REF] Song | Curcumin-loaded PLGA-PEG-PLGA triblock copolymeric micelles: Preparation, pharmacokinetics and distribution in vivo[END_REF][START_REF] Babos | Dual drug delivery of sorafenib and doxorubicin from PLGA and PEG-PLGA polymeric nanoparticles[END_REF][START_REF] Bi | Intranasal delivery of rotigotine to the brain with lactoferrin-modified PEG-PLGA nanoparticles for Parkinson's disease treatment[END_REF][START_REF] Liu | Systemic delivery of CRISPR/Cas9 with PEG-PLGA nanoparticles for chronic myeloid leukemia targeted therapy[END_REF][START_REF] Bi | T7 peptide-functionalized PEG-PLGA micelles loaded with carmustine for targeting therapy of glioma[END_REF] In a previous work, the biocompatibility of a number of PLGA-PEG copolymer micelles was studied in order to evaluate their potential in the design of drug release devices. [START_REF] Liu | Biocompatibility evaluation of self-assembled micelles prepared from poly(lactide-co-glycolide)-poly(ethylene glycol) diblock copolymers[END_REF] Various aspects of the biocompatibility were investigated, including cytocompatibility, hemocompatibility, and compatibility toward Zebrafish embryos. It was shown that PLGA-PEG micelles present good cytoand hemocompatibility in vitro, with no toxicity in vivo to embryos growth. The effects of chemical composition on micelle degradation were also evaluated. [START_REF] Su | Effects of chemical composition on the in vitro degradation of micelles prepared from poly(D,L-lactide-co-glycolide)-poly(ethylene glycol) block copolymers[END_REF]

Synthesis of PLGA-PEG copolymers

PLGA-PEG diblock copolymers were synthesized by ring opening polymerization of D,L-lactide, and glycolide in the presence of mPEG as macro-initiator and stannous octoate (Sn(Oct) 2 ) as a catalyst, as described previously. [START_REF] Su | Effects of chemical composition on the in vitro degradation of micelles prepared from poly(D,L-lactide-co-glycolide)-poly(ethylene glycol) block copolymers[END_REF] Typically, mPEG, D,Llactide, and glycolide with predetermined monomer ratios were added into a polymerization ampoule, followed by addition of stannous octoate (0.1 wt%). After several cycles of purge with nitrogen, the ampoule was sealed under vacuum.

Polymerization then proceeded at 140 ℃ for 72 h. The crude product was dissolved in dichloromethane and precipitated in cold diethyl ether. Finally, the precipitate was vacuum dried up to constant weight, yielding a copolymer in the form of a powder.

Preparation of blank micelles and drug-loaded micelles

Drug-free micelles of PLGA-PEG diblock copolymers were prepared by using the direct dissolution method. [START_REF] Su | Effects of chemical composition on the in vitro degradation of micelles prepared from poly(D,L-lactide-co-glycolide)-poly(ethylene glycol) block copolymers[END_REF] 10 mg copolymer was dissolved in 10 mL distilled water to obtain a micelle solution at a concentration of 1 mg/mL. The solution was vigorously stirred at room temperature for 3 h, followed by filtration through a 0.22 µm filter. Valsartan loaded micelles were prepared using the same method. 10 mg drug was dissolved in 400 µL tetrahydrofuran (THF), and the drug solution was dropwise added to 10 mL of previously prepared micelle solution. The ratio of drug to polymer was 20 w/w%. The mixture was vigorously stirred for 5 h to ensure total removal of the organic solvent. The resulting micelle solution was filtered through a 0.22 µm filter and centrifuged at 5000 rpm for 5 min in order to remove the unloaded drug. The supernatant was collected, freeze dried, and stored at 4 ℃ for further studies.

In vitro release of valsartan from PLGA-PEG micelles

The in vitro release of valsartan from micelles was realized by using the dialysis method.

Freeze dried valsartan-loaded micelles were dispersed in phosphate buffered saline (PBS) at a concentration of 1.0 mg/mL. 10 mL of the suspension were placed in a dialysis membrane with MWCO of 3500 Da. The samples were incubated in 70 mL PBS containing 0.1% Tween-80. Drug release was performed in water bath shaker at 37 ℃. At pre-determined time intervals, the total medium was renewed with fresh PBS so as to guarantee sink conditions.

Characterization

Proton nuclear magnetic resonance ( 1 H NMR) spectra were registered on the Bruker-Avance Ⅲ 500 Ultrashield Plus spectrometer operating at 500 MHz. Copolymer samples were dissolved in CDCl 3 at a concentration of 5 mg/mL. Chemical shifts (d)

were given in ppm, using tetramethylsilane (TMS) as internal reference.

Gel permeation chromatography (GPC) was carried out on a Waters 410 system equipped with a refractive index (RI) detector. Tetrahydrofuran (THF) was used as eluent. The sample concentration was 1.0 mg/mL. 20 µL of polymer solution were injected for each analysis. Measurements were made at 25 ℃ at a flow rate of 1 mL/min.

A calibration curve was previously established from polystyrene standards.

The size of micelles was determined by dynamic light scattering (DLS) using NanoZS90 nanosizer (Malvern, UK). Micelle solutions were prepared at a concentration of 1.0 mg/mL as mentioned above.

Transmission electron microscopy (TEM) was performed on a Hitachi-H7000 microscope (Tokyo, Japan). 5 µL of micelle solution at 1.0 mg/mL were dropped on a copper grid, and negatively stained using 1% phosphotungstic acid (PTA) solution. The Hitachi F-4600 fluorescence spectroscopy was used to determine the critical micelle concentration (CMC) of copolymers, taking pyrene was taken as a fluorescence probe. [START_REF] Jelonek | Self-assembled filomicelles prepared from polylactide/poly(ethylene glycol) block copolymers for anticancer drug delivery[END_REF] After excitation of the probe at 334 nm, the emission was recorded in the range from 350 to 450 nm. A series of copolymer solutions were prepared at concentrations from 0.0025 to 0.0125 mg/mL, whereas the concentration of the probe in solutions was fixed at 6 X10 -7 M. The CMC value were obtained from the cross-over point of the plots of the intensity ratio (I 373 /I 394 ) from pyrene emission spectra versus the logarithm of copolymer concentration.

The concentration of valsartan in solutions was measured using UV spectrophotometer.

Lyophilized micelles were dissolved in acetonitrile to disrupt the self-assembled structure. The absorbance of the solutions was measured at 288 nm using Shimadzu 2450 UV spectrophotometer (Kyoto, Japan). Drug concentration was calculated using a previously established calibration curve with a linearity range from 0.002 to 0.5 g/L (R 2 = 0.99156).

Results

Characterization of copolymers

Ring-opening polymerization of D,L-lactide, and glycolide was performed to synthesize PLGA-PEG diblock copolymers with various compositions by using mPEG and stannous octoate as macroinitiator and catalyst, respectively (Scheme 1). The molar ratios of ethylene oxide, lactyl, and glycolyl moieties (EO/LA/GA) in the feed were 4/1/1 and 6/1/1. These ratios were selected so as to ensure the solubility of copolymers in water, and thus micelles can be easily obtained by simple dissolution.

D,L-lactide, and glycolide are used to construct the degradable hydrophobic block in order to achieve appropriate degradability and drug release performance. [START_REF] Criscione | Pharmacological profile of valsartan: a potent, orally active, nonpeptide antagonist of the angiotensin II AT1-receptor subtype[END_REF] H NMR was used to determine the composition of the obtained copolymers. moieties respectively, are used to calculate the EO/LA and EO/GA ratios of copolymers.

The number average degree of polymerization (DP) of the 3 components and the Mn of the block copolymers are obtained through the following equations:

DP PEG = M nPEG /44 (1)
DP PLA = DP PEG /(EO/LA) (2)
DP PGA = DP PEG /(EO/GA) (3)
M n(NMR) = M nPEG + DP PLA ×72 + DP PGA ×58 (4) Data in parentheses correspond to feed ratios.

Self-assembly of copolymers

Critical micelle concentration

The critical micelle concentration (CMC) is a key parameter of amphiphilic block copolymers. It determines the in vivo stability of micelles after dilution by intravenous injection. The CMC of polymeric PLGA-PEG micelles is obtained by fluorescence measurements using pyrene as a probe. The emission spectra of pyrene (6 X 10 -7 M) were registered for a series of copolymer solutions at concentrations ranging from 0.0025 to 0.0125 mg/mL. With increasing the copolymer concentration of pyrene containing solutions above the CMC, noticeable changes were detected on the emission spectra due to the migration of free pyrene molecules into the hydrophobic core of micelles. The intensity ratio I 373 /I 394 was plotted against the logarithm of copolymer concentration. A significant increase in the intensity ratio is observed beyond a critical concentration, indicating the incorporation of pyrene into micelles. A CMC value is thus obtained from the cross-over point of the plots.

As shown in

Micelle size and morphology

Water soluble PLGA-PEG copolymers are allowed to self-assemble in an aqueous medium by using the direct dissolution method at a concentration of 1.0 mg/mL. The resulting micelles were characterized by using DLS and TEM. The situation is more complicated in the case of EO 114 L 30 G 27 . A network of worm-like micelles was observed, including Y-junctions and cylindrical loops, together with a number of spherical micelles. Worm-like micelles present a length of 50 to 300 nm and a diameter of 10 to 20 nm, and spherical ones exhibit a diameter of 20-40 nm (Fig. 3).

Jelonek et al. observed worm-like micelles prepared from various PLA-PEG diblock copolymers. [START_REF] Jelonek | Self-assembled filomicelles prepared from polylactide/poly(ethylene glycol) block copolymers for anticancer drug delivery[END_REF] The micelles appeared well organized with good alignment without Yjunctions. Discher et al. reported that with increasing PEG fraction, the self-assembled architecture of PLA-PEG and poly(ε-caprolactone)-poly(ethylene glycol) (PCL-PEG) block copolymers turns from solid-like particles, fluid-like vesicles, and cylinders or wormlike micelles to spherical micelles. [START_REF] Ahmed | Self-porating polymersomes of PEG-PLA and PEG-PCL: hydrolysis-triggered controlled release vesicles[END_REF] In contrast, Jain et al. observed the formation of Y-junctions which assemble into a three-dimensional network in aqueous solutions of poly(butadiene-ethylene oxide) (PB-PEO) diblock copolymers. [START_REF] Jain | On the origins of morphological complexity in block copolymer surfactants[END_REF] Y-junctions and three-dimensional network are formed above a critical molar mass at weight fractions of PEO intermediate to those associated with vesicle and worm-like micelle morphologies. On the other hand, Tlusty and Safran hypothesized that under appropriate conditions Y-junctions would appear in cylinder-forming three-component (surfactant/oil/water) microemulsions, leading to network formation. [START_REF] Tlusty | Microemulsion networks: the onset of bicontinuity[END_REF] Therefore, formation of worm-like micelles with Y-junctions for EO 114 L 30 G 27 could be assigned to its appropriate molar mass and PEG fraction as compared to the three other copolymers.

In vitro drug release

A number of factors have been identified as able to influence the drug loading capacity and encapsulation efficiency of micelles, including the core block length, architecture of micelles, drug solubility, and the drug loading conditions such as pH and temperature of the medium. [START_REF] Liu | Formulation of drugs in block copolymer micelles: drug loading and release[END_REF] The loading capacity and encapsulation efficiency of valsartan loaded micelles are listed in The partitioning of valsartan between the aqueous solution and the PLGA core determines the drug loading content. Valsartan is soluble at physiological pH with a solubility of 0.18 g/L at 25˚C. It is thus difficult to load valsartan in the hydrophobic core of micelles at physiological pH because of its good water solubility. Nevertheless, the blank micelle solution has an acidic pH of about 4 due to the presence of residual lactide/glycolide monomers which are hydrolyzed to acidic species, in agreement with previous work. [START_REF] Su | Effects of chemical composition on the in vitro degradation of micelles prepared from poly(D,L-lactide-co-glycolide)-poly(ethylene glycol) block copolymers[END_REF] Under these conditions, valsartan is unionized and has poor watersolubility and high lipophilicity. [START_REF] Thomas | Design and characterization of valsartan co-crystals to improve its aqueous solubility and dissolution behavior[END_REF] Therefore, valsartan molecules tend to migrate from the aqueous phase into the hydrophobic core of micelles.

The size and morphology of drug loaded micelles were also determined by using DLS and TEM. Fig. 4 shows the DLS graph and TEM image of EO 45 L 11 G 10 . Regularly distributed spherical micelles are detected as in the case of blank micelles. Nevertheless, the mean diameter from DLS slightly decreases from 179 nm for blank micelles to148 nm for drug loaded ones. Similar behaviors are observed in the case of EO 45 L 8 G 6 and EO 114 L 17 G 18 micelles whose size decreases from 172 to 144 nm and from 84 to 68 nm, respectively (Table 2). In fact, a micelle solution constitutes a dynamic system in which permanent exchange takes place between the micelle-forming molecules and free molecules in solution. Drug loading in the micelle core could disrupt the structure of blank micelles, resulting in the reorganization of the micellar structure and decrease of micelle size. Valsartan release from the various micellar systems was investigated under in vitro conditions. Fig. 6 shows the release profiles of valsartan in different periods up to 9 days. In general, mPEG2000 initiated copolymers exhibit faster release as compared to mPEG5000 initiated copolymers, especially in the first 24 h. An initial burst is observed in all cases. After 12 h, for example, a release ratio of 32.4, 44. respectively. A drug may be located in the core of micelles or the corona depending on its solubility characteristics and loading process. [START_REF] Allen | Nano-engineering block copolymer aggregates for drug delivery[END_REF] The initial burst could be assigned to the release of drug localized at the core-corona interface. Jelonek et al. investigated the effect of copolymer degradation on the sustained release of paclitaxel from PLA-PEG micelles in neutral and acidic media. [START_REF] Jelonek | Effect of polymer degradation on prolonged release of paclitaxel from filomicelles of polylactide/poly(ethylene glycol) block copolymers[END_REF] The authors observed that the release rate of paclitaxel strongly depends on the degradation behavior of micelles. Faster degradation leads to faster drug release. In the case of PLGA-PEG copolymer micelles, it was reported that micelles with shorter PLGA blocks degrade faster than those with longer PLGA blocks. [START_REF] Su | Effects of chemical composition on the in vitro degradation of micelles prepared from poly(D,L-lactide-co-glycolide)-poly(ethylene glycol) block copolymers[END_REF] provide longer drug release than spherical micelles. [START_REF] Geng | Shape effects of filaments versus spherical particles in flow and drug delivery[END_REF][START_REF] Jelonek | Self-assembled filomicelles prepared from polylactide/poly(ethylene glycol) block copolymers for anticancer drug delivery[END_REF] It is also of interest to examine the structure of EO 114 L 30 G 27 micelles after 7 days of drug release. As shown in Fig. 7, worm-like micelles with Y-junctions are observed, together with a number of spherical micelles. The pattern is very similar to that of blank micelles (Fig. Worm-like micelles with Y-junctions are observed, together with a number of spherical micelles.

Conclusion

Poly(D,L-lactide-co-glycolide)-poly(ethylene glycol) (PLGA-PEG) diblock copolymers with different PLGA and PEG block lengths were synthesized and characterized. The CMC of copolymers is dependent on the hydrophilic/hydrophobic balance of PEG fraction (f EO ) and the molar mass. Higher f EO and lower molar mass lead to higher CMC. The self-assembled architecture is also determined by both the hydrophilic/hydrophobic balance and the molar mass of copolymers. Different

  -N-valeryl-N-([2'-(1H-tetrazol-5-yl)-biphenyl-4-yl]-methyl)-valine, namely

  were air dried before TEM measurements.

Fig. 1 Fig. 1 .

 11 Fig. 1. 1 H NMR spectrum of PLGA-PEG diblock copolymer in CDCl 3 . The signals 1 and 2 belong to lactyl units, signal 3 to glycolyl units, signal 4 to ethylene oxide units, and signals 5, 6, and 7 to chain end and linking units.

Fig. 2

 2 Fig.2shows the DLS graphs and TEM images of micelles obtained from EO 45 L 11 G 10 , EO 45 L 8 G 6, and EO 114 L 17 G 18 . The three copolymers exhibit regular spherical structures.

Fig. 3 .

 3 Fig. 3. TEM image of blank micelles prepared from EO 114 L 30 G 27 copolymer at a concentration of 1.0 mg/ml. A network of worm-like micelles was observed, including Y-junctions, cylindrical loops, and spherical micelles.

Fig. 4 .

 4 Fig. 4. DLS graph and TEM image of valsartan-loaded EO 45 L 11 G 10 micelles (A, A') at

Fig. 5 .

 5 Fig. 5. TEM image of valsartan-loaded EO 114 L 30 G 27 micelles, showing the presence of worms and spheres.

Fig. 6 .

 6 Fig.6. In vitro release of valsartan from various micelles in 9 days (S.D. is shown as error bars, n = 3). An initial burst is observed in all cases. After 216 hours, the cumulative release from worm-like micelles is lower than that from spherical ones.

3 ).

 3 However, closer examination reveals the absence of cylindrical loops in EO 114 L 30 G 27 micelles after 7 days of drug release. The re-forming of Y-junctions could be attributed to the dynamic self-assembly of EO 114 L 30 G 27 micelles under drug release conditions. But drug release and degradation of PLGA blocks could prevent reforming of cylindrical loops.

Fig. 7 .

 7 Fig.7. TEM image of valsartan loaded EO 114 L 30 G 27 micelles after 7 days drug release.

F

  [START_REF] Yang | Novel biodegradable polylactide/poly(ethylene glycol) micelles prepared by direct dissolution method for controlled delivery of anticancer drugs[END_REF]

  Synthesis route of PLGA-PEG diblock copolymers varying opening polymerization of lactide and glycolide in the presence of monomethoxy PEG as macroinitiator and stannous octoate as catalyst.

	Scheme 1.
	F
	o r
	P
	e e r
	R e v
	i e w

Table 1

 1 are summarized the compositions of the various PLGA-PEG copolymers. The EO/LA/GA molar ratios are rather close to those in the feed, in agreement with high monomer conversion. The copolymers are named as EO x LA y GA z with x, y, and z designating the DP of ethylene oxide, lactyl, and glycolyl moieties, respectively. DP PEG is 45 for mPEG2000 and 114 for mPEG5000. DP PLA varies from 8 to 30, and DP PGA from 6 to 27. On the other hand, M n(NMR) ranges from 2924 for EO 45 L 8 G 6 to 8726 for EO 114 L 30 G 27 .The molar mass of copolymers was also determined from GPC (Table1). M n(GPC) values are in the range from 3404 for EO 45 L 8 G 6 to 8920 for EO 114 L 30 G 27 . These values are slightly superior to the M n(NMR) ones. This difference is attributed to the fact that M n(GPC) is obtained from the hydrodynamic volume of polymers in solution with respect to polystyrene standards, whereas M n(NMR) is the real molar mass determined by NMR.The dispersity (Ð = M w /M n ) of PLGA-PEG copolymers varies from 1.16 for EO 45 L 8 G 6 to 1.27 for EO 114 L 30 G 27 , which implies a narrow molar mass distribution. The weight fraction of PEG (f EO ) in the copolymers is obtained from the M n data of PEG and copolymers. It varies from 0.57 for EO 114 L 30 G 27 to 0.68 for EO 45 L 8 G 6 and EO 114 L 17 G 18 .

	F
	o r
	P
	e e r
	R e v
	i e w

Table 1 .

 1 Molecular characteristics of PLGA-PEG diblock copolymers.

	Copolymer	M n,PEG EO/LA/GA DP PEG	DP PLA	DP PGA	M n(NMR)	M n(GPC	Ð	f EO
								)
	EO 45 L 11 G 10	2000	4.5:1.1:1	45	11	10	3372	3660	1.17 0.59
			(4:1:1) a)
	EO 45 L 8 G 6	2000	7.5:1.3:1	45	8	6	2924	3400	1.16 0.68
			(6:1:1)
	EO 114 L 30 G 27 5000	4.2:1.1:1	114	30	27	8726	8920	1.27 0.57
			(4:1:1)
	EO 114 L 17 G 18 5000	6.2:0.9:1	114	17	18	7268	7420	1.25 0.68
			(6:1:1)
	a)

Table 2 .

 2

	33

b) Drug loaded micelles.

Table 2 ,

 2 the four copolymers exhibit very low CMC values of 0.0062 and 0.0077 for EO 45 L 11 G 10 and EO 45 L 8 G 6 , and of 0.0058 and 0.0065 for EO 114 L 30 G 27 and EO 114 L 17 G 18 , respectively. The data evidence the dependence of the CMC on the length of hydrophobic block or hydrophilic / hydrophobic balance (f EO ). Longer hydrophobic block or lower f EO leads to lower CMC. With similar f EO values, PEG5000 based copolymers exhibit slightly lower CMC probably due to the lower water solubility of high molar mass copolymers as reported in the literature.[START_REF] Li | Synthesis, characterization, and stereocomplex-induced gelation of block copolymers prepared by ring-opening polymerization of L (D)-lactide in the presence of poly(ethylene glycol)[END_REF]

Table 2

 2 are summarized the micelle size data derived from DLS. It appears that the diameter of EO 45 L 11 G 10 and EO 45 L 8 G 6 micelles are 179 and 172 nm, respectively. Surprisingly, EO 114 L 17 G 18 micelles exhibit a much smaller diameter of 84 nm. This finding could be explained by the fact that increasing hydrophilic block length results in stronger coronal chain repulsion. And as a consequence, the aggregation number decreases, thus leading to a smaller aggregate size.[START_REF] Cameron | EWR Steacie Award Lecture Asymmetric amphiphilic block copolymers in solution: a morphological wonderland[END_REF] On the other hand, the micelle size by DLS is much larger than that by TEM observation. This difference results from the dehydration and shrinkage of copolymer micelles during the preparation of samples for DLS graphs and TEM images of blank micelles at a concentration of 1.0 mg/ml:EO45 L 11 G 10 (A, A'), EO 45 L 8 G 6 (B, B'), and EO 114 L 17 G 18 (C, C'). DLS shows a symmetric size distribution with a mean diameter of 179, 172 and 84 nm for EO 45 L 11 G 10 , EO 45 L 8 G 6, and EO 114 L 17 G 18 , respectively. Spherical micelles are observed in all cases.

	TEM measurements.

Table 3 .

 3 Little difference is observed between the LC and EE values of EO 45 L 11 G 10 , EO 45 L 8 G 6 , and EO 114 L 17 G 18 spherical micelles despite different PLGA and/or PEG block lengths. In contrast, slightly higher EE and LC values are obtained for worm-like micelles of EO 114 L 30 G 27 copolymer as compared to spherical micelles.This difference could be attributed to the relatively larger core volume of worm-like micelles compared to spherical ones as reported in the literature.[START_REF] Cai | Micelles of different morphologies-advantages of worm-like filomicelles of PEO-PCL in paclitaxel delivery[END_REF]

Table 3 .

 3 Loading capacity and encapsulation efficiency of valsartan loaded micelles

	Copolymer	Initial drug	Loading capacity	Encapsulation
		loading (wt.%)	(wt%) a)	efficiency (%) a)
	EO 45 L 11 G 10	20	15.4±0.9	77.0±3.0
	EO 45 L 8 G 6	20	15.2±0.5	75.8±2.0
	EO 114 L 30 G 27	20	16.8±0.4	84.0±2.0

  3, 50.1, and 52.0% is detected for EO 114 L 30 G 27 , EO 114 L 17 G 18 , EO 45 L 8 G 6, and EO 45 L 11 G 10 micelles,

  Beyond 24 h, the drug release ratio continues to increase but at a slower rate. EO 45 L 8 G 6 micelles exhibit the fastest release rate with complete release after 120 h. In contrast, the lowest release rate is obtained for EO 114 L 30 G 27 micelles, 62.4% being released after 216 h. EO 114 L 17 G 18 and EO 45 L 11 G 10 micelles present intermediate release rates with 88.0 and 92.8% of the

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released drug after 216 h, respectively.

  Therefore, the different drug release rates obtained for EO 45 L 8 G 6 , EO 45 L 11 G 10 , EO 114 L 17 G 18 , and EO 114 L 30 G 27 micelles can be related to the PLGA block lengths, and consequently to the degradation rates. It is noteworthy that only 62.4% of the released drug was obtained for EO 114 L 30 G 27 wormlike micelles. This finding well agrees with literature, showing that worm-like micelles

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  architectures are observed, including spherical micelles and a mixture of spherical and worm-like micelles with Y-junctions and cylindrical loops. Valsartan is loaded in blank micelles with high encapsulation efficiency up to 84% for worm-like micelles. In vitro drug release is characterized by a burst release up to 52% in 12 h followed by slower release. The drug release rate is strongly dependent on the degradation of micelles.Copolymers with short PLGA blocks exhibit faster drug release due to faster degradation of micelles, and worm-like micelles present slower drug release as compared to spherical ones. Therefore, PLGA-PEG copolymer micelles with high drug loading capacity, different architectures and variable drug release rates could be most attractive for sustained delivery of valsartan in the treatment of hypertension.

	F F F F
	o r o r o r o r o r
	P P P P P
	e e r e e r e e r e e r e e r
	R e v R e v R e v R e v R e v
	i e w i e w i e w i e w i e w

Acknowledgment

The work was financially supported by the Talent Fund of Shandong Collaborative Innovation Center of Eco-Chemical Engineering (XTCXQN20) and the 2018 Shandong Province Graduate Education Joint Training Base Construction Project.

John Wiley & Sons

Polymers for Advanced Technologies


Supporting Information

SI Materiels and Methods

Endocytosis and recycling assays. To assay endocytosis, OP9 cells stably expressing one of the three Dll1 derivates were labelled on ice for 40 min with the reversible reagent NHS-SSbiotin (0.5 mg/mL in PBS). After a 20 min incubation at 37°C, biotin was stripped by three 25 min incubations with 50 mM MesNa (Sodium 2-mercapto-ethanesulfonate) in TNEB buffer (20 mM Tris pH 8.3; 150 mM NaCl; 1 mM EDTA; 0.2% BSA) on ice. Cells were then lysed in RIPA buffer (50 mM Tris pH 7.5, 150 mM NaCl, 1% Triton X-100, 0.1% SDS, 0.5% DOC), and biotinylated species were purified onto streptavidin-agarose and analyzed by immunoblotting with anti-Dll1CT or anti-Dll3ic antibody. To assay recycling, OP9 cells stably expressing a Dll1 derivate were labelled on ice with NHS-SS-biotin, incubated for 20 min at 37°C and biotin was stripped with MesNa as described above. Cells were incubated for 10 min on ice with 5 mg/mL iodoacetamide in TNEB. After a second incubation at 37°C for 10, 20 or 30 min, cells were treated or not with MesNa, lysed in RIPA buffer, and biotinylated species were purified onto streptavidin-agarose and analyzed by immunoblotting with anti-Dll1CT or anti-Dll3ic antibody. Immunoreactivity was quantified using Quantity One software (Bio-Rad Laboratories). For each time point, the amount of recycled ligand was estimated by substracting the amount measured in MesNa treated cells from that in non treated cells and expressed as a percentage of endocytosed ligand. Quantitation was performed after scanning of the film using Quantity One program. The percentage of biotinylated ligands at a given time point vs that at time 0 is shown.

SI figure legends

Fig. S2. Notch1 is taken up by Dll1-expressing cells. OP9 cells expressing VSV-tagged

Dll1 were co-cultured with MEFs expressing HA-tagged Notch1 for 24 hours. Both tags are in the extracellular domain of the molecules. After fixation, cells were permeabilized with detergent (a-c) or not (d-f). Dll1 was detected using a Cy3-coupled anti VSV antibody (red) and Notch1 using an Alexa488-coupled anti HA antibody (green). Hoechst staining is shown.

Overlay: superposition of the two stainings.