Julie Latune

Mariana Rios

Joana Line-Guerreiro

Eva Perrier

Géraldine Abrami

Nils Ferrand

Joana Line Guerreiro

LittoWag : a serious game to explore adaptation scenarios in front of coastal risks

niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

 French coastlines and the Occitanie region seaside (from Rhône to Spain) is vulnerable. Since 1945, 2600 hectares of land are lost, and 25% of the coastlines eroded. The Occitan coast faces strong urbanization, started in 60s, and an erosion phenomenon of the sandy coast (figure 1).  Developed coastlines are increasingly vulnerable to sea level rise, marine submersion and erosion : in 2021 the IPCC reports sea level rise between 0.16 and 0.30 meters by 2050 (table 1), and more frequent and severe storms. The uses of a model/game depend on who is involved in its design [START_REF] Etienne | Etude de l'évolution du trait de côte et des surfaces de plages du Golfe du Lion[END_REF]. It can reveal a shared problem among participants (Salliou et al. 2021). Being concerned by the problem is a prerequisite (i) to collective action [START_REF] Callon | Eléments pour une sociologie de la traduction: la-domestication-des-coquilles-saint-jacques-et-des-marins-pc3aacheurs-dans-la-baie-de-saint-brieuc[END_REF] and (ii) to use the game, and the simulation in order to learn more about the system.

Results: The LITTOWAG game Material and methods

Steps

> Ecological engineering actions are preferred to manage coastline risks (7 workshops)

Different uses of LittoWag respond to different issues depending on the stakeholders involved (figure 9): 1-A toolbox enabling those responsible for the integrated coastline management strategies to involve citizens (Occitanian regional authorities).

Figure 1 :

 1 Figure 1 : Changes in beach widths by coastal unit over several periods (data per ml/year) (EID Mediterranée, 2020)

  CreaWag method (Wat-A-Game subset) : 5 stages involved in creating a model and putting it into action: (i) framing (identification of the issues, their measurement, the intention of the model, the context of use, and the participants); (ii) construction of the conceptual model (space, issues, players, resources, activities); (iii) making the model dynamic by building a game (defining the territory, selecting roles, actions per role, resources, running a first round); (iv) finalising the game (resources dynamics); (v) calibrating the model parameters (to highlight the dilemmas, tensions, risks, induced effects and system constraints).[START_REF] Nils | WAT-A-GAME: Sharing water and policies in your own basin[END_REF][START_REF] Nils | WAT-A-GAME: Sharing water and policies in your own basin[END_REF][START_REF] Baird | Learning effects of interactive decision-making processes for climate change adaptation[END_REF]. We assess this by: -pre-post questionnaires -

  Figure 3 coastline stakes, actors, activities diagram

Figure 4 :

 4 Figure 4: LITTOWAG workshops

Figure 8 :Figure 9

 89 Figure 8: number and location of littoWag workshops

Figure

  Figure 2 : LITTOWAG content

Figure

  Figure 5 : LITTOWAG workshops

Table 1

 1

Global mean sea level projections according five global warming levels.

(IPCC, 2021)


Holger Döring
email: holgerdoering@alumni.tu-berlin.de

Space-time equation of oscillation energy in linked shell-modelanalogon of damping energy

Keywords: energy-equation, differential-equation, second-order, ftl, coupled-spacetimes, damping energy, linked shell-model, spacetime oscillation equation, Lambert-W-function, Eulerequation

The differential equation of second order for space-time-oscillation in flat tangential-spacetime of Minkowski-type can be written as an energy-equation. For this energy-equation the analogon of damping energy in two coupled Minkoswki-spacetimes with ftl can be calculated and formulated over Lamberts W-function or via Euler-equation.

1.Introduction:

The model of damped resonance can be used for special situations of spacetime description [1.], [2.].Particularly for states of oscillating spacetime. For nondamped states there is in two dimensions x , ict the system amplitude square of classical Lorentz-factor [1.], [2.]. This differential-equation (DE) can be modeled to an energy description. There can be given a solution of this description in Planck-energy form. If this equation is developed to its formal general form of a damping system, there can be derived a solution for needed damping energy of spacetime to construct ftl-states by using two local flat spacetimes [3.], [4.], [5.]. This model of ftl-state is called the "linked shell-model" because there are two local, flat Minkowski-spacetimes "linked" together over an outer damping-term like a hinge of two halfs to an image form of a closed shell [5.]. The needed Energy for this damping-state is now constructed with special-case of Planck-energy for undamped state. The damping-state-energy can be described over Lambert-function (LaF) or via Euler-equation.This will be done. Without damping-term the double-spacetimes are taken apart into two seperate states without a link and so without coupling to two ordinary flat Minkowskispaces.

2.Calculation:

The general second-order DE of space-time description is given:

A⋅ ψ+ A⋅B ψ+( A⋅C-D)⋅ψ=0
(1.) with its assigned variables of:

1. System-amplitude A : A= 1 √ (1-v ² c ² )²+ n⋅a ²⋅v ² c ⁴ (2.)
which reduces for a≡0 to squares of classical SRT-Lorentz-and Feinberg-factors [6.], [7.] and is square of advanced Lorentz-term for damped spacetime-states.The term a is damping velocity, the term of v is constant velocity of local inertial frame.

)

Then this all leads to the general equation of:

A⋅ ψ+ A⋅ W d ℏ ⋅ ψ+ ( W PL ℏ ) 2 ⋅( A-e i⋅W d ℏ ⋅t )⋅ψ=0 (7a.)
for damping states of local space-time, and with its special case for W d ≡0 to :

ℏ 2 ⋅ ψ+W PL 2 ⋅ v ² c ² ⋅ψ=0 (7b.)
for a trivial flat encoupled single spacetime (respectively the two of them) without linking, which leads straight to the right statement of:

W PL =ω PL ⋅ℏ (8.)
But the last equation of (8.) isn't surprising or a miracle because it is only that result, from what information is taken first into the equation and proves only its mathematical truthful logical quality, nothing else. In this respect, its result is a real sort of trivia, no new physics. More interesting is equation (7a.) because it allows to calculate now the energy W d , which is needed to "damp" the oscillating wavefunction of local flat space-time for causing ftl-cases of velocities for moving material bodies and link the two needed space-time states together. This calculation yields to the selfreferential equation of:

W d = ℏ⋅v r PL - W PL 2 ⋅r PL ℏ⋅v + i ⋅ W PL 2 ⋅r PL ℏ⋅A⋅v ⋅ e i W d ⋅t ℏ (9.)
The limiting case for W d ≡0 leads to the classical spacetime-case of W PL =ℏ⋅ω PL as it mustlike equation (7a.) above. So this equation (9.) for "damping energy of local spacetime" can be seen at least as mathematical logically consistent if would turn out, that it's not really physical. It should always be pointed out that the term "damping", which is in the model used, is only an analogy and does not have to correspond to the actual, real condition of the physical states because the interpretation of all these terms is not yet fully clear. There doesn't have to be a real damping. This is only a model of description. Indeed, the equation (9.) is from the selfreferential type of :

f ( x)=a+i⋅b⋅e i⋅c⋅f (x) (10a.)
This can be easily transformed into:

f ( x)=a- 1 c ⋅W (-b⋅c⋅e a⋅c ) 10b.)
and can be solved over Lamberts W-function [8.], [9.], [10.]. The solution for analogon of damping-energy therefore is then consistent with this term (10b.), which leads to the final solution for damping energy of:

W d = ℏ⋅v r PL - W ² PL ⋅r PL ℏ⋅v - ℏ i⋅t ⋅W (-1 ; 0) ( -i ⋅ W ² PL ⋅r PL ⋅t ℏ ²⋅A⋅v ⋅e i ⋅ ( ℏ⋅v r PL - W ² PL ⋅r PL ℏ⋅v ) ⋅ t ℏ ) (11a.)
where W is the Lambert-function. This term can be rewritten or rather reduced to:

W d = ℏ⋅v r PL - W PL ⋅c v +i⋅ℏ⋅Ω⋅W (-1 ; 0) ( -i ⋅ ω PL ⋅c A⋅v⋅Ω ⋅e i⋅ ( v Ω⋅r PL - ω PL ⋅c v⋅Ω ) ) (11b.)
The main question is now: when is this term (11a./11b.) real, when are there real damping energies? Instead of using the Lambert-function for this examination , it is easier to solve this problem via Euler-equation and equations (9.) and (10a.):

Equation (10a.) can be written as:

f ( x)=a-b⋅sin(c⋅f (x ))+i b⋅cos(c⋅f (x )) . (12.)
The question is, under which circumstances the imaginary cos-term in equation (12.) vanishes, to get real values. This is the case for c⋅f (x)=(2⋅n+ 1)⋅ π 2 ; n∈ℤ (13.)

Then equation (12.) reduces to:

f ( x)=a-b⋅sin(c⋅f (x )) (14.)
This equations (13.) and (14.) on the other hand means a case distinction in:

f 1 ( x )=a-b for f ( x) = (4⋅n+1) c ⋅ π 2 ; n∈ℤ (15a.)
and

f 2 ( x )=a+b for f ( x)= (4⋅n-1) c ⋅ π 2 ; n∈ℤ (15b.)
This leads finally in closer examination to equations for the physical statements of damping energies of:

W d 1 = ℏ⋅ v r PL - W ² PL ⋅r PL ℏ⋅v ⋅ ( 1+ 1 A ) for W d = ℏ t ⋅ (4⋅n+1) ⋅ π 2 ; n∈ ℤ (16a.)
and

W d 2 = ℏ⋅ v r PL - W ² PL ⋅ r PL ℏ⋅v ⋅ ( 1-1 A ) for W d = ℏ t ⋅ (4⋅n-1) ⋅ π 2 ; n∈ℤ (16b.)
This equations also can be written as:

W d , 1 =ℏ⋅Ω⋅(4 n+1)⋅ π 2 = ℏ⋅v r PL - W ² PL ⋅r PL ℏ⋅v ⋅ ( 1+ 1 A ) ; n∈ℤ (17a.)
and

W d , 2 =ℏ⋅Ω⋅(4 n-1)⋅ π 2 = ℏ⋅v r PL - W ² PL ⋅r PL ℏ⋅v ⋅ ( 1-1 A ) ; n∈ℤ (17b.)
where Ω is the damping frequency of oscillating spacetime-system.For transition to GRT there has to be taken into account that there must be distinguished between covariant and contravariant vectorfields in both coupled spacetimes.

Boundary for this energy-system can be get via solving the Lambert-function: Answer: this whole term for W d is real, when the Lambert-function

W (-1,0) =i⋅k , k ∈ℝ (18.)
This condition is (after some calculations) fulfilled for BC-terms of

v ≤√2⋅c ²-n⋅a ² ; a≤ √ 2 n ⋅c ; (19.)
as known from former papers [3.], [4.].

Remark: n=4 can be chosen from tradition of damped resonance-model for oscillating bodies. If only the term W 0 is chosen, the definition area of k is restricted to -π 2 ≤k≤ π 2 .

3.Conclusion:

The analogon of a form of "damping energy" for oscillations in Planck-area for local, flat spacetimes can be formulated. This spacetime state of damping allows coupling of two tangentialfour spaces [3.] via a link (linked shell-model of two spacetimes) like a hinge and breaks the limit of maximum local invariance velocity of c 0 for material bodies[1.], [5.] . Further informations for conditions of this case are found in [3.] Experimental measurement of effects could be difficult today, for all expected physical effects are small and leads in their limits to conditions of classical SRT, so that coupling to former theories after the modern theories of science-models are fullfilled [11.].The possibilty of existence of two solutions in W could lead to speculations over interpreting one of them as a "damping", the other as a "stimulating" process but this is not a consistent assuming and has to be proven.

Summary:

The analogon of a form of "damping-energy" for oscillating flat space-times can be calculated to get ftl-states for movements of matter with a special case without a "damping process" for getting Planck-energy. It seems, that solutions for the damping process leads possibly to mathematical complex values in ℂ , but real values are possible also which could be interpreted physically under special circumstances. These terms has to be calculated over Lambert-function W or via Euler-equation.

5.References:

[1.] Döring, H., Eigenwertproblem of advanced Lorentz-Einstein-Factor. 2023,


p. 3 nils.ferrand@inrae.fr / nils.ferrand@inria.fr • mixed agriculture with rice, irrigated, orchards and vegetables,

• petrochemical (seaside) and food industry,

• brackish laguna with RAMSAR bird area downstram • mountains & forests upstream in the natural park,

• a large multi-purpose dam, hydroelectricity, tourism .

• a shallow aquifer downstream between the city and the coast p. 14 nils.ferrand@inrae.fr / nils.ferrand@inria.fr p. 17

Field applications •

 applications France & Outre-Mer • Agence de l'Eau Rhône Méditerranée Corse : méthodologie pour des projets de participation citoyenne (PGRE, HydroMorphologie, PAPI) https://frama.link/RMCPart • Drôme (SMRD) : co-préparation citoyenne de la révision du SAGE en appui à la CLE • Camargue, Thau, StBrieuc : débat public électronique pour la GIZC • Région Occitanie : cadrage participatif citoyen de la participation en zone littorale • Luberon : dialogue sur la faisabilité sociale de la réutilisation des Eaux Usées Traitées • Nouvelle-Calédonie : planification territoriale et conception de la politique de l'eau • Europe • Bulgarie : construction participative d'un plan de cogestion inondations sécheresses • Slovénie, Suisse, Autriche, Italie : participation sur services écosystémiques rivières • Afrique, Amériques, Pacifique • Ouganda, Ethiopie, Kenya, Afrique du Sud, Mozambique, Bénin, Mali, Niger, Costa-Rica : modélisation participative, jeux de gestion, planification multi-niveaux, pour la gestion des ressources naturelles (eau, alimentation, sols, biodiversité, pollution, conflits...) • Tunisie, Brésil : (ré-)ingénierie de la gouvernance participative inter-niveaux • Sénégal : conception participative de stratégies alternatives d'assainissement • Kiribatis : modélisation participative pour aider à l'efficience du service public de l'eau p. 4nils.ferrand@inrae.fr / nils.ferrand@inria.fr COOPLAN: confronting heterogenous actions and strategies at all levels p. 5 nils.ferrand@inrae.fr / nils.ferrand@inria.fr @inrae.fr / nils.ferrand@inria.fr E.N.C.O.R.E. : coupled dimensions of change for a target group (Ferrand, le Bars, 2004) • External: external (to the group) change in sustainability • Environmental / Political / Economical • Normative: changing participants' values and preferences • Cognitive: changing participants' representations and beliefs • Operational: changing participants' practices and actions, within and outside the group and process • Relational: changing participants' social relationships • Equity: changing the social justice' regime (distribution of resources, equity) among participants and outside

  @inrae.fr / nils.ferrand@inria.frThe challenges • For the last 15 years, recharge of the dam and the river flow have clearly diminished. • Meanwhile impact of floods in the lower suburbs of the city and downstream in the touristic area near the sea got worse. • Overall carbon impact of the city is 5 time higher than rec. / IPCC • Food provision chains extends very far, with high C cost • The two factories play a key economic role, but the PetroChem plant is challenged after a pollution spillover in the river and the protected laguna. • Inequalities rise in the city and outside, with very poor farmers' communities claiming for rebalance ➔ A new development and transition plan has to be set p. 16 nils.ferrand@inrae.fr / nils.ferrand@inria.fr Phase 1 : choosing a role DO you want to be (* mandatory role in the group)? • The mayor of the city *? • The elected leader of the whole region *? • A representative of a citizens' NGO/CBO ? • An Environmental NGO representative *? • A business & real estate developer ? • The chief of the farmers' union from outside *? • A researcher in sustainability sciences ? Please take the role and endorse what you consider to be the main traits, expectations and behaviors of this.

  nils.ferrand@inrae.fr / nils.ferrand@inria.fr cc.by.nc.sa INRIA STEEP & INRAE G-EAU, 2022

1 An overall approach of M&E (Hassenforder, 2015) CONTEXT Descriptive Analytical PARTICIPATORY PLANNING PROCESS Descriptive Analytical OUTPUTS / OUTCOMES Descriptive Analytical MONITORING & EVALUATION VIEWPOINTS

		PUNCTUAL
		Attendance lists
		Pictures and videos
	Baseline studies EX-ANTE	observation Interviews Expectations Participant	EX-POST	Regular LONG TERM
	Document	Questionnaires		contacts with
	review			Interviews	the team and
	Stakeholder				key
	analysis				stakeholders
	Interviews
	Worksho	Worksho	Workshop 3 Workshop 4
	p 1	p 2	January 2013	July 2013
	April 2012	August
		2012
	PERMANE
	NT Logbook

🡪 A generic framework to observe and describe multi-dimensional change, but not to prescribe (non normative)

p. 8

nils.ferrand@inrae.fr / nils.ferrand@inria.fr Q (Based on Ferrand & Daniell, 2006; Beierle & Cayford, 2002; Midgley et al., 2013; Ostrom, 2005; Sabatier, 1988) 8 ↑ p. 9 nils.ferrand@inrae.fr / nils.ferrand@inria.fr M&E methods Q 1 ↑ p. 10 nils.ferrand@inrae.fr / nils.ferrand@inria.fr Q 1 A global M&E approach p. 11 nils.ferrand@inrae.fr / nils.ferrand@inria.fr Today's workshop: a COOPLAN starter ⇒ Issue : manage limited resources and multiple stakes in a participatory planning process toward transition ⇒ process : COOPLAN method + ENCORE evaluation ⇒ structure of the workshop : groups 5-6 persons ⇒ Case study : a large and well-off city located in a tropical region, fully dependent from its surroundings (for water, food, energy, labor, wastes) ⇒ you have to design a transition plan p. 12 nils.ferrand@inrae.fr / nils.ferrand@inria.fr Disclaimer ! This workshop simulates a participatory process held normally with any kind of stakeholders, including illiterate citizens (with support). Don't expect to engage in a complex quantitative modeling process. It's simple, robust and validated by field experience (on real case studies, not like here) It's not aimed at satisfying us, modellers, but the needs of participatory decision making. p. 13 nils.ferrand@inrae.fr / nils.ferrand@inria.fr Reasoning participation on an (abstract) case « We live in a coastal tropical region called Tropipol, in the Comoa city . Situation: • City of Comoa • (500.000 px) + demographic growth (immigration) • high GDP but high unequality • fully dependent from its surroundings for water, food, energy, waste disposal • very high individual water, energy and (virtual) soil footprint • intense touristic activity in the city and near the coast, • Outside it :

Changing what (the ENCORE+ framework with 10 dimensions) ?

	Actors' participation (process)	Expectations, preferences	Impact on natural environment
	What they know, what they believe	Behaviors, practices	Governance, political settings
	COntext
	situation around the	Social relations, ties, networks	Economic impacts
	process
		Resource
		sharing,
		equity

nils.ferrand@inrae.fr / nils.ferrand@inria.fr

Conclusion

COmments and questions welcome.


Feng Su
email: sufeng@qust.edu.cn

Yun Peng

Chenglong Li

Rongye Li

Laishun Xi

Yuandou Wang

Yangsheng Chen
email: chenys@cppqd.com

Suming Li
email: suming.li@umontpellier.fr

Novel self-assembled micelles of amphiphilic poly(2-ethyl-2-oxazoline) -poly(L-lactide) diblock copolymers for sustained drug delivery

Keywords: poly(2-ethyl-2-oxazoline), poly(L-lactide), block copolymer, self-assembly, micelles, paclitaxel, drug delivery

A series of poly(2-ethyl-2-oxazoline)-poly(L-lactide) (PEOz-PLA) diblock copolymers were synthesized by ring-opening polymerization of L-lactide using a monohydroxy terminated PEOz-OH macro-initiator in the presence of stannous octoate as catalyst. The resulting diblock copolymers were characterized by 1 H nuclear magnetic resonance and gel permeation chromatography. Self-assembled micelles were prepared using the co-solvent evaporation method in water and in phosphate buffered saline (PBS) at pH 7.4, 6.5 and 5.0. The resulting micelles exhibit different morphologies, such as spherical micelles and worm-like micelles depending on the hydrophilic/hydrophobic balance. Spherical micelles were exclusively observed for PEOz-PLA copolymers with short PLA blocks, whereas co-existence of worm-like and spherical micelles was observed for copolymers with long PLA blocks.

The micelle size increases with decreasing pH due to the electrostatic repulsion between PEOz chains resulting from ionization of the tertiary amide groups along PEOz chains. A hydrophobic anti-tumor drug, paclitaxel, was entrapped in PEOz-PLA micelles. High loading efficiency up to 86.7% was obtained for copolymers with long PLA blocks. Drug release was performed in PBS at different pH values. During the 30-day release period, faster release was obtained for copolymers with shorter PLA blocks than for copolymers with longer PLA blocks, at acidic pH than at pH 7.4. It is thus concluded that pH-responsive PEOz-PLA copolymer micellss could be promising as carrier of anti-tumor drugs.

Introduction

Paclitaxel (PTX) is a natural anti-cancer drug extracted from the bark of the pacific yew tree Taxus brevifolia. Since its approval by the U.S. Food and Drug Administration (FDA) for advanced ovarian cancer treatment in December 1992, the clinical applications of PTX have been continuously growing [START_REF] Li | Dual-functionalized nanoparticles loaded microbubbles for enhancement of drug uptake[END_REF][START_REF] Mandal | Preferential hepatic uptake of paclitaxel-loaded poly-(d-l-lactide-co-glycolide) nanoparticles -A possibility for hepatic drug targeting: Pharmacokinetics and biodistribution[END_REF]. Nowadays, PTX is considered as one of the most efficient anti-cancer drugs for the treatment of a variety of tumors, especially ovarian cancer, acellular lung cancer and metastatic breast cancer [START_REF] Meng | Use of a lipid-coated mesoporous silica nanoparticle platform for synergistic gemcitabine and paclitaxel delivery to human pancreatic cancer in mice[END_REF]. Its main mechanism of action is to promote the formation of microtubules by polymerization of tubulin dimer and to prevent depolymerization to stabilize microtubules, which results in the inability of cells to form spindle and spindle filaments during mitosis. PTX also exhibits an antitumor effect through inhibiting cell division and proliferation. Nevertheless, despite its great potential as anti-tumor drug, the clinical application of PTX is considerably restrained by its poor aqueous solubility [START_REF] Zhang | Improving paclitaxel delivery: in vitro and in vivo characterization of PEGylated polyphosphoester-based nanocarriers[END_REF]. The commonly used drug formulations of PTX are Taxol ® and Abraxane ® . The former is a co-solvent formulation composed of 50:50 Cremophor EL and dehydrated alcohol which allows to improve the bioavailability of PTX [START_REF] Bajaj | Hyaluronic acid-based hydrogel for regional delivery of paclitaxel to intraperitoneal tumors[END_REF]. The annual market of Taxol ® attained a maximum of $1.6 billion in 2000 before decreasing. In fact, serious side effects have been reported with the use of Cremophor EL, such as hypersensitivity reaction, nephrotoxicity, neurotoxicity and cardiotoxicity [START_REF] Patel | Medium chain triglyceride (MCT) rich, paclitaxel loaded self nanoemulsifying preconcentrate (PSNP): a safe and efficacious alternative to Taxol[END_REF]. Abraxane ® is an injectable formulation of albumin-bound paclitaxel nanoparticles which gained more and more importance in the past years, largely exceeding Taxol ® in clinical uses [START_REF] Chen | Drug-induced self-assembly of modified albumins as nano-theranostics for tumor-targeted combination therapy[END_REF]. However, neurotoxicity of Abraxane ® has also been reported [START_REF] De Weger | Cellular and clinical pharmacology of the taxanes docetaxel and paclitaxel--a review[END_REF].

Therefore, recent research has focused on the development of novel delivery systems of PTX, including prodrugs, emulsions, microspheres, micelles, liposomes and nanoparticles [START_REF] Zhong | Hyaluronic acid-shelled acid-activatable paclitaxel prodrug micelles effectively target and treat CD44-overexpressing human breast tumor xenografts in vivo[END_REF][START_REF] Ravar | Hyaluronic acid-coated liposomes for targeted delivery of paclitaxel, in-vitro characterization and in-vivo evaluation[END_REF]. Among them, micelles prepared by self-assembly of amphiphilic block copolymers appear most promising as drug carrier because of their outstanding properties, such as biocompatibility, nano-size, sustained drug release, high stability in vivo and in vitro, and easy binding with tumor-specific targeting ligands.

Self-assembled micelles exhibit a core-shell structure: an inner core constituted by aggregation of hydrophobic segments, and an outer shell formed by hydrogen bonding between hydrophilic segments and surrounding water molecules. The outer shell ensures stability and long circulation of the micelle system, whereas the inner core allows to encapsulate drug molecules which will be transported to the targeted site at much higher concentration than the drug's water solubility [START_REF] Almeida | Poloxamers, poloxamines and polymeric micelles: Definition, structure and therapeutic applications in cancer[END_REF][START_REF] Niu | Silica/organosilica cross-linked block copolymer micelles: a versatile theranostic platform[END_REF].

Poly(ethylene glycol) (PEG) is a FDA-approved biocompatible polyether with outstanding physico-chemical and biological properties [START_REF] D'souza | Polyethylene glycol (PEG): a versatile polymer for pharmaceutical applications[END_REF]. It's most commonly used as a hydrophilic block in amphiphilic copolymers for pharmaceutical uses. On the other hand, polylactide (PLA) is a bioresorbable and biocompatible polyester widely studied for biomedical applications [START_REF] Li | Science and Principles of Biodegradable and Bioresorbable Medical Polymers: Materials and Properties[END_REF]. PLA/PEG block copolymers are able to self-assemble to yield various aggregates, including polymersomes, worm-like or filomicelles, rod-like micelles, and spherical micelles. The morphology of aggregates mainly depends on the hydrophobic to hydrophilic balance. Filomicelles are of great interest as drug carrier due to their higher drug load and longer circulation time in the bloodstream compared to spherical micelles [START_REF] Jelonek | Self-assembled filomicelles prepared from polylactide/poly(ethylene glycol) block copolymers for anticancer drug delivery[END_REF].

Although the biomedical safety of PEG has been well admitted, it has been reported that PEG may cause complement activation in certain conditions, resulting in rapid clearance after repeated injections of PEG-containing carriers. In addition, PEG as a polyether is prone to peroxidation, thus adversely affecting cells [START_REF] Ishida | Accelerated blood clearance (ABC) phenomenon upon repeated injection of PEGylated liposomes[END_REF][START_REF] Barz | Overcoming the PEG-addiction: well-defined alternatives to PEG, from structure-property relationships to better defined therapeutics[END_REF]. Therefore, great effort has been made in the past years to search for hydrophilic alternatives of PEG.

It is known that the extracellular pH value of tumor is between 6.5 and 7.2, and that of its lysosomal/endosomal vesicles is in the range of 4.5-5.0 and 5.5-6.5. The lower pH around solid tumor tissues inspired researchers to conceive micelles which are responsive to acid-specific stimulation [START_REF] Zhao | 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]. Poly(2-ethyl-2-oxazoline) (PEOz) is a hydrophilic polymer which can be ionized at lysosomal/endosomal's pH values. (cRGDyK)-conjugated micelles for targeted delivery of paclitaxel [START_REF] Gao | PSMA-mediated endosome escape-accelerating polymeric micelles for targeted therapy of prostate cancer and the real time tracing of their intracellular trafficking[END_REF][START_REF] Gao | Enhanced antitumor efficacy by cyclic RGDyK-conjugated and paclitaxel-loaded pH-responsive polymeric micelles[END_REF].

Besides

Recently, Wang et al. investigated the mechanisms of pH-sensitivity and cellular internalization of a series of PEOz-PDLLA micelles with various hydrophilic/hydrophobic ratios [START_REF] Wang | Mechanisms of pH-sensitivity and cellular internalization of PEOz-b-PLA micelles with varied hydrophilic/hydrophobic ratios and intracellular trafficking routes and fate of the copolymer[END_REF]. The authors suggested that electrostatic repulsion between PEOz chains resulting from ionization of the tertiary amide groups along PEOz chain at pH lower than its pKa was responsible for pH-sensitivity of PEOz-PDLLA micelles. Nevertheless, few studies focused on the self-assembly behavior of PEOz-PLLA copolymers under different pH conditions. In fact, it has been shown that the morphology of self-assembled micelles is dependent on the hydrophilic/hydrophobic ratio, block length, and chain structure regularity of copolymers [START_REF] Jelonek | Self-assembled filomicelles prepared from polylactide/poly(ethylene glycol) block copolymers for anticancer drug delivery[END_REF]. Various micelle structures, including spherical, rod-like and worm-like micelles can be obtained from PEG-PLA, PEG-PCL, and PEG-P(CL/GA) copolymers, which strongly affects the drug release behavior and blood circulation time of micelles [START_REF] Jelonek | Self-assembled filomicelles prepared from polylactide/poly(ethylene glycol) block copolymers for anticancer drug delivery[END_REF][START_REF] Sun | Self-assembled micelles prepared from poly(ɛ-caprolactone)-poly(ethylene glycol) and poly(ɛ-caprolactone/glycolide)-poly(ethylene glycol) block copolymers for sustained drug delivery[END_REF].

In this paper, a series of PEOz-PLLA copolymers were synthesized by ring-opening polymerization of L-lactide, using hydroxyl-terminated PEOz-OH as macro-initiator and stannous octoate as catalyst. Self-assembled micelles of the copolymers were prepared using co-solvent evaporation method and characterized. The drug loading and drug release properties of PTX-loaded PEOz-PLA micelles were evaluated, considering the influence of copolymer composition and pH value. The results are reported herein in comparison with literature.

Materials and methods

Materials

L-lactic acid was obtained from Sigma (St Louis, MO, USA). L-lactide was synthesized according to literature method, and purified by recrystallization three times from ethyl acetate [START_REF] Zhang | Preparation of monomer of degradable biomaterial poly(L-lactide)[END_REF].

Synthesis of PEOz-PLA copolymers

Amphiphilic PEOz-PLA diblock copolymers were synthesized as reported in literature [START_REF] Lee | Synthesis and micellar characterization of amphiphilic diblock copolymers based on poly(2-ethyl-2-oxazoline) and aliphatic polyesters[END_REF]. Typically, 2-ethyl-2-oxazoline (20.0 g, 200 mmol) and methyl p-toluene sulfonate (1.24 g, 6.67 mmol) were added to dry acetonitrile (60 mL), and the temperature was gradually increased to 100 °C. The reaction then proceeded at reflux for 30 h under nitrogen atmosphere. After cooling down to room temperature, 0.1 M potassium hydroxide in methanol was added to the solution, and the reaction continued 4 more hours to introduce hydroxyl groups. The crude product was filtered through silica gel, and the filtrate was added to cold diethylether. The precipitated product PEOz-OH was dried under vacuum for 24 h.

Secondly, PEOz-OH (1.0 g), stannous octoate (5 mg), L-lactide (0.58 g, 4 mmol), and chlorobenzene (15 mL) were introduced in a Schlenk flask. The reaction proceeded at 120 °C for 30 h under N2 atmosphere. The product was filtered through silica gel, precipitated by addition of diethylether and finally dried under vacuum up to constant weight. And the yield of the final product is about 90%.

Preparation of polymeric micelles and drug-loaded micelles

PEOz-PLA micelles were prepared by using co-solvent evaporation method [START_REF] Rajagopal | Curvature-coupled hydration of semicrystalline polymer amphiphiles yields flexible worm micelles but favors rigid vesicles: polycaprolactone-based block copolymers[END_REF].

Briefly, 20 mg copolymer was dissolved in 1 mL chloroform. The solution was slowly added to 20 mL phosphate buffered saline (PBS) at pH 7.4, and stirred for 4 h to allow self-assembly of micelles along with solvent evaporation. The resulting micelle solution was filtered through 0.8 µm filter and stored at 4 °C.

PTX-loaded micelles were prepared using a two-step procedure according to the literature [START_REF] Sun | Self-assembled micelles prepared from poly(ɛ-caprolactone)-poly(ethylene glycol) and poly(ɛ-caprolactone/glycolide)-poly(ethylene glycol) block copolymers for sustained drug delivery[END_REF]. Blank micelles were first prepared as mentioned above. 1 mg PTX dissolved in 1 mL methanol was then added to 10 mL blank micelle solution. The solution was vigorously stirred for 4 h, and gently stirred for 24 h to complete solvent evaporation. The solution was finally centrifuged at 3 000 rpm for 10 min to remove unloaded drug. The supernatant was collected as drug-loaded micelle solution.

Characterization

1 H nuclear magnetic resonance (NMR) spectra were recorded at room temperature on a Bruker AVANCE III 500 spectrometer (Billerica, MA, USA) operating at 500 MHz, using CDCl3 as solvent. Chemical shifts (δ) were given in ppm using tetramethylsilane as an internal reference.

Gel permeation chromatography (GPC) measurements were performed on a Shimadzu apparatus (Kyoto, Japan) equipped with a Waters 410 refractometer and a PLgel 5μm MIXED-C column. Tetrahydrofuran (THF) was used as the mobile phase at a flow rate of 1.0 mL/min. The sample solution at a concentration of 1.0 mg/mL was injected for analysis. Polystyrene standards were used for calibration.

Dynamic light scattering (DLS) was realized on a Nano-ZS90 nanosizer (Malvern, Worcestershire, UK). Measurements were performed at 25 °C with a 90° scattering angle.

Transmission electron microscopy (TEM) was performed on JEM-1200EX microscope (JEOL, Japan). A small amount of micelle solution in PBS was dropped on a carbon coated copper grid. Then the excess fluid was removed with a filter paper, followed by staining using 1.0 wt.% phosphotungstic acid solution and air drying.

Examination of micelles was realized at an acceleration voltage of 80 kV.

The critical micelle concentration (CMC) of PEOz-PLA copolymers was measured by fluorescence spectrophotometer (F-4600) using pyrene as fluorescent probe [START_REF] Dominguez | Determination of critical micelle concentration of some surfactants by three techniques[END_REF].

Briefly, 1 mL pyrene solution (2 × 10 -5 M in benzene) was added to 10 mL volumetric flask, and the solvent was evaporated. Different volumes of micelle solutions were added to the flask. Then distilled water was added to a total volume of 10 mL. The resulting micellar concentrations ranged from 1.0×10 -3 to 0.5 mg/mL, and the final concentration of pyrene was 2.0×10 -6 M. After equilibrium at room temperature for 24 h, the fluorescence excitation spectra of the micellar solutions were recorded from 350 to 450 nm at an excitation wavelength of 334 nm. The intensity ratio at 375 nm and 395 nm was plotted versus copolymer concentration. The CMC value was obtained from the cross-over point of two regression lines.

High-performance liquid chromatography (HPLC) was performed using a Waters HPLC (Milford, MA, USA) equipped with a UV detector and a C18 column. The detection wavelength was set at 227 nm. The mobile phase was a mixture of water and acetonitrile (v/v 45/55). The column temperature was 30 °C. 10 µL solution was injected for each analysis at a flow rate of 1.0 mL/min.

In vitro release of PTX from PEOz-PLA micelles

PTX release from PEOz-PLA micelles was realized under in vitro conditions. Briefly, 3 mL drug-loaded micelle solution was introduced in a cellulose dialysis bag (molecular weight cutoff 3500) which was then placed in 30 mL PBS containing 0.1% Tween 80. The pH of the drug release media was 7.4, 6.5, or 5.0, respectively. Drug release was performed at 37 °C in a water bath shaker at 80 rpm. At pre-determined time points, 1 mL of the release medium was removed, and replaced with the same volume of medium. The content of PTX in the release medium was determined by using HPLC.

Results and discussion

Characterization of PEOz-PLA copolymers

Amphiphilic PEOz-PLA block copolymers were synthesized by ring-opening polymerization of L-lactide as illustrated in Fig. 1. Hydroxyl-terminated PEOz-OH was first synthesized by polymerization of 2-ethyl-2-oxazoline using methyl tosylate as initiator, followed by chain termination with methanolic KOH. The hydroxyl endgroup of PEOz-OH was used to initiate the ring-opening polymerization of L-lactide in the presence of stannous octoate as catalyst, yielding PEOz-PLA diblock copolymers with different PEOz and PLA block lengths. respectively. The [LA/EOz] ratios are slightly lower than those in the feed as shown in Table 1, suggesting that the conversion of L-lactide is not complete. In fact, the yield of the reactions was 80 to 90%. The DPPLA and the number-average molar mass (Mn) of the copolymers are then obtained from the following relationships: DPPLA = DPPEOz x [LA/EOz] (eq. 1)

Mn = DPPEOz x 99 + DPPLA x 72 (eq. 2)

Where 99 and 72 are the molar of EOz and LA repeat units, respectively.

The compositions of the various copolymers are summarized in Table 1. The copolymers are named as EOzxLy for the sake of clarity. In this acronym, EOz and L represent PEOz and PLLA blocks, respectively, while x and y designate the number-average degree of polymerization of corresponding blocks. The DPPLA varies from 42 to 6 for the EOz28Ly series, with Mn varying from 5820 to 3230 g/mol. And a copolymer with composition of EOz18L5 and Mn of 2160 was also synthesized.

The Mn and dispersity (Ð = Mw/Mn) of the copolymers are also determined from GPC measurements, as summarized in Table 1. The Mn(GPC) values are close or slightly superior to Mn(NMR) ones. In fact, the Mn(GPC) is determined with polystyrene standards and is usually higher than those from NMR, as reported in literature [START_REF] Sun | Self-assembled micelles prepared from poly(ɛ-caprolactone)-poly(ethylene glycol) and poly(ɛ-caprolactone/glycolide)-poly(ethylene glycol) block copolymers for sustained drug delivery[END_REF]. On the other hand, the dispersity varies from 1.47 for EOz28L42 to 1.18 for EOz18L5, in agreement with narrow molar mass distributions.

The hydrophilic-lipophilic balance (HLB) is a major parameter of amphiphilic polymers which can be determined by Griffin's method. A low HLB value indicates strong hydrophobicity, and vice versa. Table 1 shows that the HLB varies from 9.6 for EOz28L42 to 17.3 for EOz28L6. Obviously samples with higher PEOz content have a higher HLB, in agreement with higher hydrophilicity.

Table 1

Structural characteristics of PEOz-PLLA diblock copolymers.

a [LA/EOz] is calculated from NMR, and data in parentheses are the feed ratios.

b HLBcopolymer = (MnPEOz/Mncopolymer)×20.

Self-assembly of copolymers

Critical micelle concentration

The CMC is an essential parameter that determines the behavior of micelles in vivo, especially in terms of micelle stability after intravenous injection. The CMC of PEOz-PLA copolymers was obtained from fluorescence spectrophotometer using pyrene as the hydrophobic probe. The fluorescence emission spectra of pyrene were registered in the range of 350 to 450 nm with increasing copolymer concentration from 1.0×10 -3 to 0.5 mg/mL (Fig. S1). The emission intensity of samples at low concentrations is almost the same, but a significant increase occurs when the concentration reaches a critical value. The intensity ratio I375/I395 vs. logC changes of micelles were plotted, and the crossover point of the two regression lines is taken as the CMC (Fig. S1). The CMC data of the various copolymers are shown in Table 1. It appears that the CMC is closely related to the PLA block length. The longer the hydrophobic PLA block, the lower the CMC value. In fact, amphiphilic block copolymers with longer hydrophobic blocks are more inclined to self-assemble in aqueous medium, thus leading to lower CMC and higher structural stability [START_REF] Liggins | Polyether-polyester diblock copolymers for the preparation of paclitaxel loaded polymeric micelle formulations[END_REF]. It is noteworthy that the CMC values of all samples are very low (0.004-0.008 mg/mL) compared to low molar mass surfactants, which should ensure the stability of micelles after administration in the bloodstream [START_REF] Zhu | Spontaneous generation of amphiphilic block copolymer micelles with multiple morphologies through interfacial instabilities[END_REF].

Morphology of self-assembled structures

Micelle size and morphology strongly affect the drug loading and drug release properties. PEOz-PLA micelles were prepared by self-assembly in pH 7.4 PBS using co-solvent evaporation method. The morphology of PEOz-PLA blank and PTX-loaded micelles was examined by TEM as shown in Fig. 3. Both spherical and worm-like micelles are observed for EOz28L42 (Fig. 3A). The spherical micelles exhibit a diameter of 20-50 nm, and the worm-like micelles a length of about 200 nm and a diameter of about 30 nm. In contrast, uniformly dispersed spherical micelles are observed for EOz28L21 (Fig. 3B). The TEM images of micelles after PTX loading are shown in Fig. 3A' and Fig. 3B'. Obviously drug loaded micelles exhibit the same structures as blank micelles. Similar results are obtained in the case of EOz28L10, EOz28L6 and EOz18L5 micelles (Fig. S3). Thus copolymers with HLB value above 13.0 only yield spherical micelles. DLS was used to determine the size and size distribution of spherical micelles in aqueous solution (Fig. S2). A uniform micelle size distribution is observed in all cases.

The effect of pH on the micelle size was investigated as PEOz exhibits some pH-sensitivity. The various copolymers were allowed to self-assemble in water and in PBS at pH = 7.4, 6.5, 5.0 by using co-solvent evaporation method, and characterized by using DLS. All the micelle data are summarized in Table 2. Data of EOz28L42

are not available as it forms both spherical and worm-like micelles. For the two poorly water-soluble copolymers (EOz28L21 and EOz28L10), the diameter of micelles decreases with decreasing PLA block length. In fact, the size is 103.6 nm for EOz28L21 and 74.6 nm for EOz28L10, which is in agreement with literature [START_REF] Wang | Mechanisms of pH-sensitivity and cellular internalization of PEOz-b-PLA micelles with varied hydrophilic/hydrophobic ratios and intracellular trafficking routes and fate of the copolymer[END_REF]. This finding could be explained by the increased aggregation number of copolymer micelles with longer PLA blocks which attracted more copolymer chains in a micelle.

It is also of interest to note that the diameter obtained from TEM is much lower than that from DLS, which can be attributed to the dehydration and shrinkage of micelles during TEM measurements.

On the other hand, it appears that the size of micelles self-assembled in water is smaller than that in PBS, the micelle size increases with decreasing pH. These findings could be related to the presence of tertiary amide bonds in PEOz structure. In a weakly acidic environment, the tertiary amide bond allows adsorption of protons, resulting in the formation of PEOz intrachain or interchain hydrogen bonds.

Meanwhile, the electrostatic repulsion between PEOz chains resulting from ionization of the tertiary amide groups along PEOz chain also contributes to micelle size increase with decreasing pH. The situation is more complicated in the case of water-soluble copolymers (EOz28L6 and EOz18L5). In contrast to kinetically "frozen" micelles obtained from water insoluble copolymers, EOz28L6 and EOz18L5 copolymer micelles are dynamic systems with permanent exchanges between micelle-forming molecules and free molecules in solution, continuously breaking and reforming [START_REF] Yang | Micelles formed by self-assembling of polylactide/poly(ethylene glycol) block copolymers in aqueous solutions[END_REF].

Surprisingly, little difference of micelle size was observed in water and in PBS at different pH values probably because of the dynamic nature of micelles which allows self-adjustment of micelle architecture and size [START_REF] Wang | Mechanisms of pH-sensitivity and cellular internalization of PEOz-b-PLA micelles with varied hydrophilic/hydrophobic ratios and intracellular trafficking routes and fate of the copolymer[END_REF][START_REF] Sun | Self-assembled micelles prepared from poly(ɛ-caprolactone)-poly(ethylene glycol) and poly(ɛ-caprolactone/glycolide)-poly(ethylene glycol) block copolymers for sustained drug delivery[END_REF]. It is also noted that the polydispersity index of micelles ranges from 0.21 to 0.35 in all cases.

Table 2

Diameter of blank micelles in different media and loading efficiency (LE) and loading content (LC) data of PTX-loaded micelles.

a Dh and PDI of micelles determined by DLS.

b LE and LC determined by HPLC. Data represent mean value ± S.D., n = 3.

In vitro drug release

PTX was used as a hydrophobic model drug to evaluate the drug loading and release properties of PEOz-PLA copolymers. Drug loading was realized by using a two-step procedure consisting in adding drug solution into a pre-formed micelle solution under stirring. The drug loading content (LC) was defined as the ratio of the amount of loaded drug to the total amount of drug loaded micelles, and the loading efficiency (LE) as the ratio of the amount of loaded drug to the amount of initially introduced drug (theoretical drug loading) according to the following equations [START_REF] Yang | Novel biodegradable polylactide/poly(ethylene glycol) micelles prepared by direct dissolution method for controlled delivery of anticancer drugs[END_REF][START_REF] Jelonek | Effect of polymer degradation on prolonged release of paclitaxel from filomicelles of polylactide/poly(ethylene glycol) block copolymers[END_REF]:

LE = weight of loaded drug theoretical drug loading × 100% (1)
LC = weight of loaded drug weight of drug-loaded micelles × 100% Considering the overall performance of the loading content, loading efficiency and release rate, EOz28L21 and EOz28L10 micelles were used to evaluate the effect of pH on drug release. Three pH values (7.4, 6.5, 5.0) were used to mimic the physiological pH, extracellular pH of tumor, and that of lysosome/endosome of tumor. Fig. 4B shows the PTX release profiles from EOz28L21 and EOz28L10 copolymer micelles at different pH values. Drug release appears rather smooth without burst effect. The release ratio from EOz28L21 is 11.6% at pH 7.4, 14% at pH 6.5, and 20.9% at pH 5.0 in 30 days. Similarly, the release ratio from EOz28L10 is 19.4% at pH 7.4, 23.2% at pH 6.5, and 29.4% at pH 5.0. These findings indicate that the release rate is only slightly enhanced at pH 6.5, and significantly enhanced at pH 5.0 as compared to pH 7.4. It has been reported that the pKa of PEOz-PLA was 6.5 [START_REF] Wang | Mechanisms of pH-sensitivity and cellular internalization of PEOz-b-PLA micelles with varied hydrophilic/hydrophobic ratios and intracellular trafficking routes and fate of the copolymer[END_REF]. Thus the hydrophilic PEOz block could be ionized at pH 5.0 which is below its pKa, leading to swelling of micelles and increase of micelle size which should accelerate drug release.

In fact, protonation of amide groups is an intermediate step of the acid hydrolysis of poly(2-alkyl-2-oxazoline)s (PAOx) to obtain polyethylenimine (PEI) [START_REF] Mees | Full and partial hydrolysis of poly(2-oxazoline)s and subsequent post-polymerization modification of the resulting polyethylenimine (co)polymers[END_REF]. During the hydrolysis process, the amide group is activated by protonation of the carbonyl It is also noteworthy that acidic environments could enhance the degradation of PLA blocks, which could also accelerate drug release. However, it has been reported that the cumulative release of PTX from PEG-PLLA micelles at pH 5.5 is only 2.5% higher than that of pH 7.4 in 30 days, and the difference of release rates between pH 7.4 and pH 3.0 was less than 3% [START_REF] Jelonek | Effect of polymer degradation on prolonged release of paclitaxel from filomicelles of polylactide/poly(ethylene glycol) block copolymers[END_REF]. Thus it can be assumed that the pH-responsiveness of PEOz-PLLA micelles is the main reason for the enhanced drug release at acidic pH.

It has been reported that PEOz with higher molar mass may have higher pH-responsive effect [START_REF] Wang | New amphiphilic poly(2-ethyl-2-oxazoline)/poly(L-lactide) triblock copolymers[END_REF]. Thus the release profiles of PTX from EOz28L6 and EOz18L5 micelles were comparatively studied, as shown in Fig. 4C. The release ratio from EOz28L6 and EOz18L5 is 36% and 37.6% at pH 5.0, respectively. This finding could be attributed to the small difference of molar masses between the two samples.

It is also noted that 25.8% drug release is obtained for EOz28L6 at pH 7.4, which is much lower than that at pH 5.0, in agreement with the enhanced drug release at acidic pH.

The release rate of PTX from of PEOz-PLLA micelles appears much slower as compared to the results reported in literature [START_REF] Wang | Mechanisms of pH-sensitivity and cellular internalization of PEOz-b-PLA micelles with varied hydrophilic/hydrophobic ratios and intracellular trafficking routes and fate of the copolymer[END_REF]. In fact, Wang et al. reported that PTX release from the micelles was pH dependent and markedly accelerated with decreasing pH value. 67% of PTX were released within 48 h from PEOz6-b-PLA4 micelles with Mn of PEOz of 6000 and Mn of PEOz of 4000, whereas PTX release was 75% and 90% at pH 6.5 and 5.0, respectively. The differences could be assigned to the chemical composition and release conditions. In the present work, 0.1% Tween 80 was added in the release media, in contrast to 0.2% Tween 80 in literature. Higher Tween content should enhance drug release as it improves drug solubility. On the other hand, PLLA block used in this study has lower degradability than PDLLA block used in literature. And faster degradation of the polyester block favors drug release [START_REF] Jelonek | Effect of polymer degradation on prolonged release of paclitaxel from filomicelles of polylactide/poly(ethylene glycol) block copolymers[END_REF]. Last but not least, as PLLA degrades very slowly in PBS at pH 7.4, 6.5 and 5.0 [START_REF] Jelonek | Effect of polymer degradation on prolonged release of paclitaxel from filomicelles of polylactide/poly(ethylene glycol) block copolymers[END_REF], drug molecules are more likely to be released through diffusion from the interior of PEOz-PLLA micelles.

It is also of interest to compare the self-assembly and drug release behaviors of PEOz-PLLA micelles and other micelle systems [START_REF] Jelonek | Self-assembled filomicelles prepared from polylactide/poly(ethylene glycol) block copolymers for anticancer drug delivery[END_REF][START_REF] Sun | Self-assembled micelles prepared from poly(ɛ-caprolactone)-poly(ethylene glycol) and poly(ɛ-caprolactone/glycolide)-poly(ethylene glycol) block copolymers for sustained drug delivery[END_REF][START_REF] Yang | Micelles formed by self-assembling of polylactide/poly(ethylene glycol) block copolymers in aqueous solutions[END_REF][START_REF] Yang | Novel biodegradable polylactide/poly(ethylene glycol) micelles prepared by direct dissolution method for controlled delivery of anticancer drugs[END_REF][START_REF] Jelonek | Effect of polymer degradation on prolonged release of paclitaxel from filomicelles of polylactide/poly(ethylene glycol) block copolymers[END_REF]. Various micelle structures, including spherical, rod-like and worm-like micelles have been observed from PEG-PLLA, PEG-PDLLA, PEG-PCL, and PEG-P(CL/GA) copolymers, depending on the hydrophilic/hydrophobic ratio, block length, and chain structure regularity of copolymers. In particular, spherical micelles are exclusively obtained for PEG-PDLLA copolymers, whereas both spherical and worm-like micelles are formed from PEG-PLLA copolymers [START_REF] Jelonek | Self-assembled filomicelles prepared from polylactide/poly(ethylene glycol) block copolymers for anticancer drug delivery[END_REF]. Jelonek et al. studied PTX release from various PEG-PLA micelles. The LE of all samples ranges from 48% to 69%, and LC from 4.7% to 6.5%, which is comparable to PEOz-PLLA micelles. Nevertheless, less than 25% of drug was released during 71 days [START_REF] Jelonek | Self-assembled filomicelles prepared from polylactide/poly(ethylene glycol) block copolymers for anticancer drug delivery[END_REF]. On the other hand, little difference was found between the release rates of PEG-PLLA micelles at pH 7.4, 5.5 and 3.0 during the first 5 weeks [START_REF] Rosa | Fast and accurate partial hydrolysis of poly(2-ethyl-2-oxazoline) into tailored linear polyethylenimine copolymers[END_REF]. Therefore, pH-sensitive PEOz-PLLA micelles could be a promising carrier of hydrophobic anti-tumor drugs.

Conclusions

In this work, amphiphilic PEOz-PLA copolymers were successfully synthesized by ring-opening polymerization and considered as a potential drug carrier for controlled delivery of anti-tumor drugs. Self-assembled micelles were prepared using co-solvent evaporation method in water and in PBS at pH 7.4, 6. Faster release was obtained for copolymers with shorter PLA blocks than for copolymers with longer PLA blocks, at acidic pH than at pH 7.4. It is thus concluded that pH-responsive PEOz-PLA copolymers could be promising as carrier of anti-tumor drugs. Further studies are underway in terms of micelle morphology, drug release, in vitro cytotoxicity, and in vivo cellular uptake, in particular the differences between spherical and worm-like micelles.

  , PEOz presents outstanding biocompatibility, which makes it a lactide)-poly(2-ethyl-2-oxazoline)-poly(L-lactide) (PLLA-PEOz-PLLA) triblock copolymers, and investigated the polymers' pH-sensitive properties as potential drug carrier [19]. Gulyuz et al. prepared PEOz-PLA and poly(2-ethyl-2-oxazoline)-poly(ɛ-caprolactone) (PEOz-PCL) diblock copolymers, and evaluated their cytotoxicity in vitro [22]. Zhao et al. studied the release of doxorubicin (DOX) from poly(2-ethyl-2-oxazoline)-poly(D,L-lactide) (PEOz-PDLLA) micelles. Data showed that the release of DOX was faster at pH 5.0 than at pH 7.4 [18]. Wang et al. studied the in vitro release, cellular uptake and in vitro cytotoxicity of PEOz-PDLLA micelles co-encapsulating PTX and honokiol (HNK) [23]. The dual drug-loaded PEOz-PLA micelles exhibited small size and high drug encapsulation efficiency, and favorable pH-dependent drug release characteristics. Qiu et al. attached folic acid to PEOz-PCL, and investigated the cellular internalization and anticancer activity of DOX-loaded micelles of FA-PEOz-PCL [24]. Wang et al. synthesized PEOz-PDLLA-PEOz triblock copolymers. The resulting hydrogels showed good cytocompatibility in vitro [25]. Gao et al. synthesized HOOC-PEOz-PDLLA to prepare YPSMA-1-modified micelles and cyclic RGDyK

Fig. 1 .

 1 Fig. 1. Synthesis route of PEOz-PLA copolymers.

Fig. 2 .

 2 Fig. 2. 1 H-NMR spectra of PEOz-OH (A) and PEOz-PLA copolymer (B).

Fig. 3 .

 3 Fig. 3. TEM images of PEOz-PLLA blank micelles (left) and PTX-loaded micelles (right): EOz28L42 (A, A'), EOz28L21 (B, B').

Fig. 4 .

 4 Fig. 4. (A) In vitro release profiles of paclitaxel from EOz28L42, EOz28L21, EOz28L10 and EOz28L6 copolymer micelles at pH = 7.4 PBS at 37 °C; (B) In vitro release profiles of paclitaxel from EOz28L21 and EOz28L10 copolymer micelles at pH = 7.4, 6.5, 5.0 PBS at 37 °C; (C) In vitro release profiles of paclitaxel from EOz28L6 and EOz18L5 copolymer micelles at pH = 7.4 and pH = 5.0 PBS at 37 °C. All data are represented as the mean ± SD (n = 3).

  functional group, followed by formation of a tetrahedral intermediate via nucleophilic attack of water. Finally the carboxylic acid is detached from the polymer backbone to yield PEI. It should be noticed that amide groups are very stable and hydrolysis of PAOx only occurs under strong acidic or basic conditions [39-41]. In the present work, protonation of the amide group in PEOz chain well agrees with the variation in micelle size and release rate at various pH values.

  5 and 5.0. The resulting micelles exhibit different morphologies, such as spherical micelles and worm-like micelles depending on the hydrophilic/hydrophobic balance. The micelle size increases with decreasing pH due to the electrostatic repulsion between PEOz chains resulting from ionization of the tertiary amide groups along PEOz chains. The PTX-loading and release properties of micelles were also closely related to the length of PLA. Copolymers with longer PLA segments present higher LE and LC values.

Table 2

 2 presents the LC and LE data of PTX-loaded micelles. EOz18L5 exhibits the lowest LE of 40.6% and LC of 3.9%, whereas EOz28L21 presents the highest LE of 86.7% and LC of 8.0%. Obviously the LC and LE values mainly depend on the length of the hydrophobic block of copolymers. The longer the hydrophobic block, the higher LE and LC values of copolymer micelles, and vice versa. However, lower LE and LC values are obtained for EOz28L42 as compared to EOz28L10 and EOz28L21. In fact, EOz28L42 has a high hydrophobic PLA content or low HLB value. Thus the micelles tend to aggregate and precipitate during the self-assembly process, leading to lower drug loading efficiency in spite of the co-existence of spherical and worm-like micelles.It is also of interest to compare the released drug amounts from different micelle systems. For example, EOz28L21 exhibits a release percentage of 11.6% in 30 days, but it has a high loading efficiency of 86.7%. In contrast, EOz28L6 presents the highest release rate (25.8% in 30 days), but its loading efficiency is only 43.5%. In 30 days, the total released drug from EOz28L21 is approximately 0.030 mg, and that from EOz28L6 is about 0.034 mg.

	Fig. 4A presents the drug release profiles from 4 micelle samples (EOz28L42,
	EOz28L21, EOz28L10 and EOz28L6) with different PLA block lengths in pH 7.4 PBS at
	37 °C. No burst release is detected. It appears that the PTX release ratio is improved
	for copolymers with short PLA blocks. EOz28L6 exhibits the highest release ratio with
	25.8% of released drug in 30 days, in contrast to EOz28L42 which has only 6.5% drug
	release. EOz28L10 and EOz28L21 exhibit intermediate release ratios of 19.4% and
	11.6%, respectively. EOz28L42 with low drug content shows the lowest release rate.
	This finding could be assigned to the most compact core structure of EOz28L42
	micelles which disfavors drug diffusion to the medium.

Acknowledgement

The work is supported by the Science and Technology Development Plan of Shandong Province (2018GGX102016), and the Qingdao Municipality Science & Technology Huimin Plan (16-6-2-17-nsh).


Feng Su

Xiangke Sun

Rongye Li

Yuandou Wang

Laishun Xi

Yangsheng Chen

Suming Li
email: suming.li@umontpellier.fr

Drug release and biocompatibility of self-assembled micelles prepared from poly(ɛ-caprolactone/glycolide)-poly(ethylene glycol) block copolymers

Keywords: micelle, poly(ɛ-caprolactone), poly(ethylene glycol), paclitaxel, drug release, biocompatibility

Poly(ɛ-caprolactone/glycolide)-poly(ethylene glycol) (P(CL/GA)-PEG) diblock copolymers were prepared by ring opening polymerization of a mixture of ɛ-caprolactone and glycolide using monomethoxy PEG as macro-initiator and Sn(Oct)2 as catalyst. The resulting copolymers were characterized by using 1 H NMR and GPC. Self-assembled micelles were prepared from the copolymers using nanoprecipitation method. The morphology of micelles was spherical as revealed by TEM. The micelle size was larger for copolymers with longer PEG blocks, and the CMC of copolymers increased with decreasing the overall hydrophobic block length.

Drug loading and in vitro drug release studies were performed using paclitaxel as a hydrophobic model drug. Higher drug loading was obtained for micelles with longer PCL blocks. Faster drug release was obtained for micelles of mPEG2000 initiated copolymers than those of mPEG5000 initiated ones. Higher GA content in the copolymers led to faster drug release. Moreover, drug release rate was enhanced in the presence of a lipase, indicating that drug release is facilitated by copolymer degradation. Furthermore, copolymer micelles present outstanding biocompatibility as evidenced by hemolysis, dynamic clotting time and plasma recalcification time tests, as well as MTT assay and agar diffusion test, thus suggesting that P(CL/GA)-PEG micelles are promising for prolonged release of hydrophobic drugs.

INTRODUCTION

Paclitaxel, as an anti-microtubule drug, has demonstrated efficient anticancer activity against various tumors [1][2][3] . Paclitaxel behaves as promoter of tubulin's assembly and inhibitor of its disassembly, and thus restrain cell mitosis 4,5 . Unfortunately, clinically used formulation of paclitaxel containing Cremophor EL can accelerate the release of histamine and cause serious adverse events including neutropenia, allergic reaction, neurotoxicity, cardiovascular toxicity, gastrointestinal reaction, liver and gallbladder reaction, etc 5 . Furthermore, conventional cancer chemotherapy using paclitaxel presents no selectivity towards cancer cells and normal cells, thus producing obvious side effects to normal tissues [START_REF] Sparreboom | Anti-cancer drugs[END_REF] . Therefore, finding a safer and more effective paclitaxel delivery system without Cremophor EL becomes a major issue in the treatment of cancers.

In the past decades, aliphatic polyesters such as polylactide (PLA), poly(ε-caprolactone) (PCL), poly(glycolide) (PGA) and their copolymers have become the focus of biodegradable materials due to their excellent biodegradability and biocompatibility [START_REF] Chiellini | [END_REF] . In the biomedical field, they are widely used as surgical sutures, controlled drug release devices and tissue engineering scaffolds. These polyesters can degrade by hydrolysis into small molecules which are then eliminated from the body through natural pathways. The physico-chemical properties of polyesters can be tailored by introducing a biocompatible and hydrophilic component such as poly(ethylene glycol) (PEG) 8,9 . The resulting amphiphilic block copolymers including PLA-PEG, PCL-PEG, poly(lactide-co-glycolide)-PEG (PLGA-PEG) and poly(ɛ-caprolactone/glycolide)-PEG (P(CL/GA)-PEG) have received more and more attention in drug delivery systems [10][11][12][13] . In fact, they can self-assemble to form micelles with a core-shell structure in aqueous solution: a hydrophobic core able to encapsulate hydrophobic drugs, and an outer hydrophilic shell ensuring biocompatibility and long circulation in blood. Moreover, the size of micelles (< 200 nm) is small enough to avoid being swallowed by the immune system or filtering out by the liver and spleen [14][15][16] .

Biocompatibility is a key factor for clinical applications of biomaterials. It refers to the complex biological, chemical and physical interactions between material and human body, and the extent of endurance of receptors to these reactions. In the case of drug carriers, they should not cause any undesirable local or systemic effects in the recipient or host of therapy 17 . Although the biocompatibility of PCL, PGA and PEG polymers has been well documented, very few data are available on the biocompatibility of copolymer micelles in aqueous environment in vitro.

In our previous work, we comparatively studied the self-assembly, drug loading and drug release properties of PCL-PEG and P(CL/GA)-PEG micelles as novel carrier of paclitaxel 18 . The morphology of micelles is mainly dependent on the hydrophilic/hydrophobic balance of copolymers and the regularity of hydrophobic chain structure. Worm-like or filomicelles were obtained for PCL-PEG copolymers in all cases. In contrast, spherical micelles were obtained with addition of a small amount of GA component which disrupts the chain structure. Drug release proceeded very slowly in all cases. P(CL/GA)-PEG micelles presented slightly faster release than PCL-PEG micelles, but the overall release rate didn't exceed 10% in 30 days. Therefore, it is of major importance to enhance the drug release from P(CL/GA)-PEG micelles by increasing the proportion of GA component.

In the present work, a series of P(CL/GA)-PEG diblock copolymers with various EO/(CL/GA) molar ratios were synthesized by ring opening polymerization of a mixture of ɛ-caprolactone and glycolide in the presence of monomethoxy poly(ethylene glycol) (mPEG) as macroinitiator. P(CL/GA)-PEG micelles were prepared by co-solvent evaporation method. Drug loading was performed using paclitaxel as model drug, and drug release was studied under in vitro conditions in a pH 7.4 phosphate buffered saline (PBS). The biocompatibility of P(CL/GA)-PEG micelles was evaluated from the aspects of hemocompatibility and cytocompatibility in order to assess the micelles' potential as drug delivery systems.

EXPERIMENTAL

Materials

ε-Caprolactone was obtained from J&K (Beijing, China), and purified by distillation.

Glycolide was purchased from J&K and purified by crystallization from ethyl acetate.

Monomethoxy PEG (mPEG) with molar masses of 2000 or 5000 was supplied by Sigma-Aldrich (Shanghai, China). Stannous octoate (Sn(Oct)2) and paclitaxel were supplied by Aladdin (Shanghai, China). All organic solvents were of analytic grade.

Synthesis of P(CL/GA)-PEG Copolymers

P(CL/GA)-PEG copolymers were synthesized by ring-opening polymerization of ε-caprolactone and glycolide initiated by mPEG in the presence of Sn(Oct)2 as catalyst as previously reported (Scheme 1) 19 . Briefly, predetermined amounts of mPEG, ε-caprolactone, glycolide and Sn(Oct)2 were added into a polymerization tube.

The mixture was degased, and then sealed under vacuum. Polymerization then proceeded at 130℃ for 2 d. The resulting product was recovered by dissolution in dichloromethane and precipitation with diethyl ether, followed by vacuum drying up to constant weight.

Preparation of P(CL/GA)-PEG micelles

Micelles were prepared using a solvent evaporation process as previously described 20 .

Briefly, 20 mg copolymer was dissolved in 400 μL chloroform, and then 20 mL distilled water was added. The solution was stirred vigorously at room temperature for 3.5 h, and allowed to stay 24 h for solvent evaporation. The resulting micellar solution was filtered through 0.45 μm pore-sized syringe filter. The concentration of the final micellar solution was 1.0 mg/mL.

Characterization

Proton nuclear magnetic resonance ( 1 H NMR) was employed to determine the composition of copolymers. The spectra were recorded on Bruker AVANCE Ⅲ 500 spectrometer operating at 500 MHz, using CDCl3 as a solvent. Chemical shifts (δ) were given in ppm using tetramethylsilane as an internal reference.

The molecular weight and molecular weight distribution of copolymers were determined by gel permeation chromatography (GPC) carried out on a Shimadzu apparatus equipped with a Waters 410 refractometer. Tetrahydrofuran was used as mobile phase at a flow rate of 1.0 mL/min. 60 μL copolymer solution at a concentration of 1.0 mg/mL was injected for analysis. Polystyrene standards were used for calibration.

Dynamic light scattering (DLS) was used to measure the size and size distribution of micelles, using a Nano-ZS90 nanosizer (Malvern, UK) equipped with a digital time correlator. The concentration of micellar solutions was 1.0 mg/mL, and measurements were made at 25℃ with a scattering angle of 90°.

The critical micelle concentrations (CMC) of P(CL/GA)-PEG micelles were determined by spectrofluorimeter (Hitachi F-7000), using pyrene as fluorescence probe 21 . Breifly, 1 mL pyrene solution (2 × 10 -5 M in benzene) was added to 10 mL volumetric flask, and the solvent was evaporated. Different volumes of micelle solutions were added to the flask. Then distilled water was added to a total volume of 10 mL. The resulting micellar concentrations ranged from 9.7×10 -4 to 0.5 mg/mL, and the final concentration of pyrene was 2.0×10 -6 M. After equilibrium at room temperature for 24 h, the fluorescence excitation spectra of the micellar solutions were recorded from 350 to 450 nm at an excitation wavelength of 334 nm. The excitation and emission slit widths were 5 nm and 2.5 nm, respectively, and the scanning speed was 500 nm/min. The excitation ratios of I375/I395 were used for the determination of the CMC value.

The morphology of micelles was examined by using transmission electron microscopy (TEM). Measurements were realized on JEM-1200EX microscope, operating at a high voltage of 80 kV. The carbon coated copper grid was immersed in a micellar solution of 1.0 mg/mL for 1 min, and then taken out. One drop of 3% phosphotungstic acid (PTA) solution was placed on the copper grid for negative staining, followed by air drying at room temperature before measurements.

In vitro drug release

Paclitaxel loading in micelles was performed by using solvent evaporation method 22 .

Briefly, 2 mg paclitaxel was dissolved in 1 mL methanol, and then dropped into 10 mL pre-formed micelle solution under constant stirring at room temperature. The solution was stirred overnight to allow paclitaxel encapsulation. Unloaded paclitaxel was removed by centrifugation at 5,000 rpm for 10 min.

High performance liquid chromatography (HPLC) was used to determine the amount of paclitaxel in drug loading and drug release experiments. A WATERS-e2695 apparatus equipped with a C18 column (4.6 × 250 mm, pore size 5 μm, WATERS-e2695, America) was used, and the detection wavelength of UV detector was set at 227 nm. A mixture of HPLC grade acetonitrile-water (v/v, 55/45) was used as mobile phase at a flow rate of 1.2 mL/min. The drug loading content (LC) was defined as the ratio of the amount of loaded drug to the total amount of drug loaded micelles, and the loading efficiency (LE) as the ratio of the amount of loaded drug to the amount of initially introduced drug 3 .

Drug release experiments were realized under in vitro conditions. 10 mL drug loaded micelle solution was introduced into a dialysis membrane (MWCO=3500), and immersed in 10 mL PBS at pH 7.4. Drug release was then conducted at 37℃ with constant shaking. At predetermined time intervals, the release medium was totally withdrawn for analysis, and the same amount of fresh PBS was added.

Hemocompatibility evaluation

Hemolysis tests were realized in accordance with ISO 10993-4:2009 and ASTMF756-00 [START_REF]4: Selection of tests for interactions with blood[END_REF][START_REF]Standard Practice for Assessment of Hemolytic Properties of Materials [S[END_REF] , using fresh acid citrate dextrose (ACD) anticoagulated rabbit whole blood (blood/3.8wt% citrate acid = 9:1, V/V). 10 mL micellar solution at 1 mg/mL was used as test group, 10 mL distilled water and 0.9% saline water as positive and negative controls. All tubes were placed in a water bath thermostated at 37℃ for 0.5 h, and 0.2 mL diluted blood (blood/saline=4:5, V/V) was then added.

After 1 h, all samples were centrifuged at 3000 rpm for 5 min at room temperature, and the OD value of the supernatants was measured at 545 nm by using UV-visible spectrophotometer (T6-1650F, China). All experiments were repeated three times (n = 3). The hemolysis ratio (HR) was calculated according to following formula 25 :

    100 ) /( (%) negative positive negative test     OD OD OD OD HR
Where ODtest, ODnegative, and ODpositive are the absorbance values of the micellar solution, negative and positive controls, respectively.

The dynamic clotting time was determined according to the literature 26 . 1 mg/mL micellar solution was prepared in 0.2 M CaCl2 solution, and 10 μL the above solutions were used as test group. Siliconized and non-siliconized glass tubes containing 10 μL 0.2 M CaCl2 solution were used as negative and positive controls, respectively. All tubes were thermostated in a 37℃ water bath for 5 min. Then 80 μL ACD blood was added into tubes. After 0, 10, 20, 40, 60, 80, 100, 120 min, 20 mL deionized water was gently added. The OD values of the supernatants were measured at 490 nm using a microplate reader. The relative clotting time for each micellar solution was obtained from the OD versus time plots. All experiments were repeated five times (n = 5).

Plasma recalcification time (PRT) was measured by using a literature method 25 .

Platelet poor plasma (PPP) was acquired by centrifugation of fresh ACD anticoagulated whole blood at 3000 rpm for 10 min. Micellar solutions at 1 mg/mL were prepared in 0.025 M CaCl2 solution, and 100 μL of the above solutions were used as test group. 100 μL of 0.025 M CaCl2 solution were added into siliconized and non-siliconized glass tubes, and used as negative and positive controls, respectively. All tubes were thermostated in a 37 ℃ water bath for 2 min, and then 100 μL PPP was added. The tube was tilted every 2 s to observe the clotting condition of PPP after 50 s.

The time of clotting was recorded at the moment when plasma solution could no longer flow. All experiments were repeated three times (n = 3).

Cytocompatibility evaluation

Micellar solutions at 1 mg/mL were prepared in growth medium (DMEM containing 10% fetal bovine serum), and diluted to various concentrations of 0.05, 0.1, 0.2, 0.5 mg/mL. MTT assay was employed to evaluate the cytotoxicity of micellar solutions in vitro 27,28 , using L-929 cells in logarithmic growth phase. Cell suspensions were prepared with growth medium to a concentration of 1 × 10 4 cells / mL, and 100 μL cell suspension was seeded in 96-well plates (Corning Costar, USA) placed in 5% CO2 incubator (NU-4850; NuAire, USA) at 37 ℃ under humidified environment.

After 24 h incubation, the medium was removed and replaced by 100 μL micellar solution. 100 μL fresh medium was used as the negative control, and 100 μL 6.4% phenol aqueous solution as the positive control. After incubation for 1, 2 and 3 days, 20 μL MTT solution at 5 mg / mL was added. The medium in the well was removed after 4 h incubation, and then 150 μL DMSO was added. After 10 min shaking, the optical density (OD) was measured at 490 nm by using microplate reader (Elx800, BioTek, USA). All samples were measured three times (n=3). The relative growth rate (RGR) was determined by using the following equation:

100 ) / ( (%) group   control negative test

OD OD RGR

Agar diffusion test was realized according to the literature method [START_REF] Liu | Polymers for Advanced Technologies[END_REF] , using L-929 cells in logarithmic growth phase. The cells were diluted with growth medium (DMEM containing 10% fetal bovine serum) to a concentration of 2.5 × 10 4 cells / mL. 10 ml cell suspension was placed in a Petri dish and cultured in 5% CO2 incubator at 37 ℃. After 24 h, the culture medium was discarded, and the dish washed with PBS. 12 mL of 1.5% fresh agar medium were then added. When the agar medium was completely coagulated, 10 ml neutral red solution was added. The neutral red solution was removed after 15-20 min. 100 μL micellar solution was dropped into a piece of filter paper (diameter 5 mm) which was carefully placed on a dish. After exposure at 37 ℃ in a 5% CO2 humidified atmosphere for 24 h, the toxicity was assessed by inverted microscopy from the width of the decolorization zone around the test solution and sloughing or lysis within the zone. 100 μL fresh medium and 6.4% phenol aqueous solution were used as the negative and positive controls, respectively.

Statistics analysis

Statistics analysis was performed using SPSS 10.0 software. Descriptive data were expressed as the arithmetic mean value plus or minus the standard deviation. All quantitative results were obtained from at least triplicate samples. A value of p < 0.001 was considered to be extremely significant, p < 0.01 very significant, p < 0.05 statistically significant, and p > 0.05 not significant.

Results and discussion

Characterization of Copolymers

A series of 6 P(CL/GA)-PEG diblock copolymers were obtained by ring-opening polymerization of a mixture of CL and GA monomers, using monomethoxy PEG with Mn of 2000 or 5000 as macroinitiator and Sn(Oct)2 as catalyst. The initial CL/EO feed ratio was 0.5, and the CL/GA ratio was 1, 2 or 5. The various copolymers were named as P(CL/GA)-PEG2K X or P(CL/GA)-PEG5K X, where 2K and 5K represent the molecular weight of the PEG block and X the CL/GA ratio.

The composition of the copolymers was determined by 1 H NMR [START_REF] Mutlu | [END_REF] . Fig. 1 shows the

Mn = MnPEG + DPPCL×114 + DPPGA×116 (3)
Table 1 summarizes the molecular characteristics of the various P(CL/GA)-PEG copolymers. The DPPEG is 45 or 114 for mPEG2000 and mPEG5000, respectively.

The DPPCL ranges from 24 to 26 for mPEG2000 initiated copolymers, and from 52 to 62 for mPEG5000 initiated copolymers. Moreover, the DPPGA ranges from 5 to 20 for mPEG2000 initiated copolymers, and from 12 to 65 for mPEG5000 initiated copolymers. In all cases, the compositions of the resulting copolymers are close to those of the feeds, which is consistent with a good conversion of monomers. In fact, all copolymers were obtained with high yields above 80%. Concerning the Mn, it ranges from 5540 to 7060 for mPEG2000 initiated copolymers, and from 13460 to 18450 for mPEG5000 initiated copolymers.

The molecular weights and dispersity (Đ=Mw/Mn) of P(CL/GA)-PEG copolymers were also determined by GPC. As shown in Table 1, the Mn(GPC) ranges from 6250 to 8180 for mPEG2000 initiated copolymers, and from 15310 to 19620 for mPEG5000 initiated copolymers. In fact, the Mn(GPC) values were obtained with respect to polystyrene standards, and are generally higher than the absolute molecular weights determined by NMR. The dispersity (Đ= Mw/Mn) values of all copolymers range from 1.3 to 1.5, indicating a narrower distribution of molecular weights. a Data in parentheses correspond to the feed ratio.

Self-assembly of P(CL/GA)-PEG copolymers

Micelles of P(CL/GA)-PEG copolymers were prepared using a solvent evaporation process. The morphology of micelles was examined by means of TEM. As shown in Fig. 2, micelles of P(CL/GA)-PEG2K 5 exhibit a spherical shape with a diameter of about 25 nm. Similar spherical shapes are observed for other copolymers. The average particle size and polydispersity index (PDI) were determined by means of DLS. As shown in Table 2, the micelle diameter is 136.8, 121.3 and 104.7 nm for P(CL/GA)-PEG2K 1, P(CL/GA)-PEG2K 2 and P(CL/GA)-PEG2K 5, respectively.

This finding shows that the micelle size decreases with decreasing hydrophobic block length (DPPCL+DPPGA). Meanwhile, a diameter of 158.6, 147.2, and 132.8 nm is obtained for P(CL/GA)-PEG5K 1, P(CL/GA)-PEG5K 2 and P(CL/GA)-PEG5K 5, respectively. These findings suggest that the micelle size increases with increasing length of hydrophilic PEG segments in the copolymer, which is consistent with literature data 32 . The PDI of micelle size is in the range from 0.251 to 0.272, in agreement with narrow size distribution. It is also of interest to note that the diameter obtained from TEM is much lower than that from DLS, which can be attributed to the dehydration and shrinkage of micelles during TEM measurements 33 . As a key parameter that characterizes the stability of micelles, the CMC of diblock copolymers was evaluated by using fluorescent probe method. Fig. 3(A) shows the emission spectra of pyrene at different micellar concentrations. An increase in the fluorescence intensity and a red shift are observed with increasing concentration. Fig.

3(B)

shows the intensity ratio (I375/I395) vs. log C plots of P(CL/GA)-PEG2K 1. The CMC value was obtained from the cross-over point of the regression lines. As shown in Table 2, all copolymers present very low CMC values (2.4-6.8 μg/mL), which should be beneficial for micellar stability after intravenous administration and dilution in bloodstream 34 . These values are comparable to those of other copolymers that generally present very low CMC values ranging from 1 mg/mL to 10 mg/mL 35 , and are much lower than typical CMC values of low molecular weight surfactants 36 . It is also noteworthy that copolymers with longer hydrophobic blocks exhibit lower CMC values for both mPEG2000 and mPEG5000 initiated copolymers, in agreement with higher micelle stability as reported in literature 34 . Jelonek et al. reported that the CMC of PLA-PEG diblock copolymers decreases as the length of hydrophobic PLA segment increases. In fact, copolymers with longer hydrophobic blocks present a lower CMC as they can more easily self-assemble to form micelles 35 .

Paclitaxel release from P(CL/GA)-PEG micelles

Paclitaxel loading in micelles was realized by employing solvent evaporation method.

Drug loading content (LC) and loading efficiency (LE) data of mPEG2000 and mPEG5000 initiated copolymers are summarized in Table 2. The loading content is 8.8%, 9.8%, and 11.4% for P(CL/GA)-PEG2K 1, P(CL/GA)-PEG2K 2, and P(CL/GA)-PEG2K 5, respectively. These findings could appear surprising as copolymers with longer hydrophobic blocks should lead to higher drug loading according to literature 34 , and P(CL/GA)-PEG2K 1 has the longest overall hydrophobic block among the three mPEG2000 initiated copolymers. It is thus assumed that micelles with longer PCL block length lead to higher drug loading.

Similar findings were reported for micelles prepared from PEG-b-P(CL-co-TMC) diblock copolymers 37 . Latere et al. found that higher PCL content in the copolymers tends to enhance the solubility of furosemide. The drug loading properties of mPEG5000 initiated copolymers are slightly better than those of mPEG2000-initiated ones, which is in accordance with the literature data 22 . The loading content is 9.2%, 10.6%, and 12.8% for P(CL/GA)-PEG5K 1, P(CL/GA)-PEG5K 2, and P(CL/GA)-PEG5K 5, respectively.

Fig. 4 and 5 present the release profiles of paclitaxel from the various micellar systems in pH =7.4 PBS at 37°C. The drug release proceeds slowly for all copolymer micelles without burst release. However, some differences are noticed for the various micellar systems. 25.3%, 23.2% and 20.8% of paclitaxel release are obtained for P(CL/GA)-PEG2K 1, P(CL/GA)-PEG2K 2, P(CL/GA)-PEG2K 5 in 30 days, respectively. This indicates that higher PGA content in the copolymers leads to faster paclitaxel release. In fact, faster degradation of micelles leads to faster drug release [38]. With the increase of PGA component, the disruption of the chain structure and chain cleavage would be enhanced as in the case of PLGA copolymers 38,39 , thus improving the drug release rate. Similarly, 16.8%, 14.8% and 13.2% of released paclitaxel were obtained for micelles of P(CL/GA)-PEG5K 1, P(CL/GA)-PEG5K 2 and P(CL/GA)-PEG5K 5 after 30 days, respectively. Thus paclitaxel release is faster for mPEG2000 initiated copolymers than for mPEG5000 initiated ones. This finding could be attributed to the larger micelle size, and to the larger hydrophobic core of mPEG5000 initiated copolymer micelles with longer hydrophobic blocks. Both disfavors drug diffusion. Similar findings have been reported for PLA-PEG copolymer micelles 34 .

Drug release was also studied in pH=7.4 PBS containing lipase from Pseudomonas sp.

to evaluate the effect of enzymatic degradation on drug release rate. This enzyme is known to accelerate the degradation of PCL based polymers 9 , although it is not present in the body. Faster paclitaxel release is observed in the presence of enzymes in all cases. For example, 25.3% of drug release were detected for P(CL/GA)-PEG2K 1, in contrast to 32.0% of drug release for P(CL/GA)-PEG2K 1E (E referring to enzymes). Similarly, 16.8% of released paclitaxel were obtained for micelles of P(CL/GA)-PEG5K 1, whereas 23.8% were found for P(CL/GA)-PEG5K 1E.

Therefore, the presence of lipase from Pseudomonas sp. enhanced the drug release rate P(CL/GA)-PEG micelles. It is also of interest to note that in our previous work, paclitaxel release rate is below 10% for micelles prepared from PCL-PEG and P(CL/GA)-PEG with CL/GA ratio of 10 18 . This further confirms that drug release can be improved by enhancing copolymer degradation by incorporation of faster degrading PGA component.

Hemocompatibility evaluation

Hemolysis test

Hemolysis, which demonstrates the disturbed integrity of the membrane structure of red blood cells (RBC), is determined by the release of intracellular hemoglobin.

Hemolysis test is widely used in vitro to evaluate the biocompatibility of biomaterials 40 . The hemolysis ratio (HR) indicates the degree of hemolysis and intracellular hemoglobin dissociation when a biomaterial is in contact with RBC in vitro. The higher the HR value, the higher the breaking open degree of RBC. HR values below 5% indicate good hemocompatibility and vice versa 41 .

The HR of the different micellar solutions was obtained from the OD values as shown in Table 3. The HR values of all micelles are lower than 5%. These findings suggest that all micelles have little effect to the erythrocytes, indicating the outstanding anti-hemolysis properties of micelles.

Dynamic clotting time

Dynamic clotting time experiments are often employed to assess the activated degree of endogenous coagulation factors. There is a close relationship between endogenous coagulation process and blood coagulation occurrence. Endogenous coagulation occurs when the micelle is in contact with blood, and the degree of blood clotting gradually increases as the contact time increases. The absorbance-time curve reflects the trend of blood clotting and the duration of clotting time 42 . The higher the absorbance value, the better the anticoagulation properties. It is generally admitted that the initial clotting time is reached when the absorbance is reduced to 0.1, and the blood is completely coagulated when the absorbance drops to 0.01.

As shown in Fig. 6, the dynamic clotting time curves of micellar solutions are similar to that of the negative control, showing a slowly descending trend. In contrast, the positive control exhibits a faster decrease of the OD value. The initial clotting time of P(CL/GA)-PEG2K 1, P(CL/GA)-PEG2K 2, P(CL/GA)-PEG2K 5, P(CL/GA)-PEG5K The first stage of blood coagulation happens through the endogenous and exogenous pathways, leading to the formation of prothrombinase. There exists a cascade of reactions between coagulation factors 43 . Plasma recalcification time (PRT) represents the time required for fibrin clot formation when Ca 2+ is added into PPP. It is thus an important indicator of micelle induced coagulation activation 44 .

The longer the PRT, the better the hemocompatibility of biomaterials. The PRT is 374.7 , 417.0, 435.3, 397.8, 428.6 and 452.4 s for P(CL/GA)-PEG2K 1, P(CL/GA)-PEG2K 2, P(CL/GA)-PEG2K 5, P(CL/GA)-PEG5K 1, P(CL/GA)-PEG5K

2 and P(CL/GA)-PEG5K 5, respectively (Fig. 7). These values are slightly smaller than that of the negative control (479.6 s), but significantly larger than that of the positive control (243.3 s), thus showing that the micelles present good hemocompatibility.

Cytocompatibility evaluation

MTT assay

MTT assay is one of the preferred methods to evaluate the cytocompatibility of biomaterials [START_REF] Luo | Polymers for Advanced Technologies[END_REF] . L-929 cell line (mouse fibroblast) was used for MTT test as it is a commonly used standard cell line in cytocompatibility evaluation.

The relative activity of cells in the presence of micellar solutions is shown in Fig. 8. It appears that the cell viability in the positive control is very low, while the micellar solutions have little effect on the growth of L-929 cells as well as the negative control group (100%). The cell viability slightly varies with the incubation time and micellar concentration. The cell viability slightly decreases (P > 0.05) with the increase of micellar concentration from 0.05 to 1.0 mg/mL,but the incubation time up to 72 h doesn't influence the viability in all groups (P >0.05). It is worth noting that even with the highest concentration of 1 mg/mL and at the longest time of 72 h, the relative activity is above 80% for all micellar solutions. All these data show that P(CL/GA)-PEG micellar solutions are not toxic to L-929 cells.

Agar diffusion test

Agar diffusion test is another method of choice used to evaluate the cytotoxicity of biomaterials according to ISO 10993-5 [START_REF] Schmalz | [END_REF] . It is based on the fact that neutral red preferentially acts on acid regions of living cells such as proliferating DNA and lysosomes, thus dyeing them red 47 . Therefore, the toxicity of P(CL/GA)-PEG micellar solutions is related to the decolorization caused by undyed damaged cells and cell injury morphological signs 48 . As shown in Fig. 9

CONCLUSIONS

In this work, P(CL/GA)-PEG diblock copolymers with various compositions were obtained by ring-opening polymerization of ε-caprolactone (CL) and glycolide (GA) using mPEG2000 or mPEG5000 as initiator and Sn(Oct)2 as catalyst. The effect of copolymer composition on the micellar size, critical micelle concentration, drug loading and drug release properties was elucidated. All the micelles are spherical in shape, and the micelle size is larger for copolymers with longer PEG blocks. In contrast, the CMC of copolymers increases with decreasing the overall hydrophobic block length. The drug loading is closely related to the length of PCL blocks and B C copolymer composition. Higher drug loading is obtained for micelles with longer PCL blocks. Drug release is mainly dependent on copolymer degradation. Micelles prepared from mPEG2000 initiated copolymers exhibit faster release than those of mPEG5000 initiated copolymer micelles, and higher GA content leads to faster paclitaxel release. Moreover, drug release rate is enhanced in the presence of lipase from Pseudomonas sp., thus indicating that drug release is favored by copolymer degradation.

On the other hand, the hemocompatibility and cytocompatibility of micelles were evaluated. Low hemolytic ratio, low effect on the dynamic clotting time and plasma recalcification time indicate good hemocompatibility of the various micellar solutions.

MTT and agar diffusion tests suggest that all micellar solutions present very low cytotoxicity on cell adhesion and proliferation. Therefore, these bioresorbable P(CL/GA)-PEG micelles, with excellent stability, high drug loading content, prolonged drug release and good biocompatibility could be promising for applications as drug carrier.

Scheme 1 .

 1 Scheme 1. Synthesis route of P(CL/GA)-PEG copolymers

1 H

 1 NMR spectrum of P(CL/GA)-PEG2K 5. The signal at 3.63 ppm (b) is attributed to the methylene groups of PEG block, whereas the signals at 4.07 (c), 2.31 (f), 1.65 (d) and 1.38 ppm (e) are assigned to the methylene groups of PCL moieties. The signal in the 4.6-4.8 ppm zone (g) belongs to the methylene group of PGA block. Besides, two smaller downfield signals are detected beside those at 4.07 (c) and 2.31 (f). These new signals are attributed to the caprolactone units which are linked to glycolidyl units in the P(CL/GA) block. These findings demonstrate that P(CL/GA)-PEG diblock copolymers were successful obtained 31 .

Figure 1 . 1 H

 11 Figure 1. 1 H NMR spectrum of P(CL/GA)-PEG diblock copolymers

Figure 2 .

 2 Figure 2. TEM image (left) and DLS graph (right) of P(CL/GA)-PEG2K 5 micelles.

Figure 3 .

 3 Figure 3. (A) Fluorescence emission spectra of pyrene with increasing concentrations of P(CL/GA)-PEG2K 1. (B) Plots of the intensity ratios (I375/I395) versus the concentration of P(CL/GA)-PEG2K 1 copolymer.

Figure 4 .Figure 5 .

 45 Figure 4. In vitro release profiles of paclitaxel in pH =7.4 PBS and pH =7.4 PBS with lipase.from mPEG2000-initiated copolymer micelles (S.D. shown as error bars, n=3)

Figure 6 .

 6 Figure 6. Curves of dynamic clotting time of micelles in comparison with the controls

Figure 7 .

 7 Figure 7. Plasma recalcification time of micelles. ** indicates p < 0.01, *** p < 0.001, and * p > 0.05.

Figure 8 .

 8 Figure 8. Relative activity of L-929 cells after 24 h (A), 48 h (B), and 72 h (C) culture with micelle solutions at different concentrations as compared to the negative

AFigure 9 .

 9 Figure 9. Microscopic images of L-929 cells after 24 h exposure to the negative control (A), P(CL/GA)-PEG2K 1 (B) and positive control (C) in agar diffusion test.

Table 1 .

 1 Structural characteristics of P(CL/GA)-PEG block copolymers

	Copolymer	MnPEG	CL/EO a	CL/GA a	DPPEG	DPPCL	DPPGA	Mn(NMR)	Mn(GPC)	Đ
	P(CL/GA)-PEG2K 1	2000	0.53 (0.5)	1.2 (1)	45	24	20	7060	8180	1.41
	P(CL/GA)-PEG2K 2	2000	0.56 (0.5)	1.8 (2)	45	25	14	6480	7200	1.45
	P(CL/GA)-PEG2K 5	2000	0.58 (0.5)	5.7 (5)	45	26	5	5540	6250	1.39
	P(CL/GA)-PEG5K 1	5000	0.46 (0.5)	0.8 (1)	114	52	65	18450	19620	1.36
	P(CL/GA)-PEG5K 2	5000	0.51 (0.5)	1.5 (2)	114	58	39	16140	17260	1.51
	P(CL/GA)-PEG5K 5	5000	0.54 (0.5)	5.2 (5)	114	62	12	13460	15310	1.40

Table 2

 2

		. Characterization of P(CL/GA)-PEG diblock copolymer micelles
	copolymer	Size (nm) a	PDI a	CMC(mg/mL)	LE(%) a	LC(%) a
	P(CL/GA)-PEG2K 1 136.8±12.7 0.253±0.02	0.0024	52.8±4.3 8.8±0.6
	P(CL/GA)-PEG2K 2 121.3±10.2 0.256±0.03	0.0042	58.8±5.7 9.8±0.8
	P(CL/GA)-PEG2K 5 104.7±11.4 0.251±0.05	0.0048	68.4±6.4 11.4±0.9
	P(CL/GA)-PEG5K 1 158.6±14.6 0.272±0.04	0.0036	55.2±8.6 9.2±1.2

P(CL/GA)-PEG5K 2 147.2±12.4 0.264±0.07 0.0040 63.6±7.6 10.6±0.7 P(CL/GA)-PEG5K 5 132.8±10.8 0.258±0.08 0.0068 76.8±10.2 12.8±1.4 a Data represent mean value ±S.D., n = 3.

Table 3 .

 3 Hemolysis ratios of different copolymer micelles

	Copolymer	OD Value	Hemolysis ratio (%)
	P(CL/GA)-PEG2K 1	0.048±0.005	2.9±0.8
	P(CL/GA)-PEG2K 2	0.052±0.004	3.3±0.7
	P(CL/GA)-PEG2K 5	0.042±0.007	2.2±0.8
	P(CL/GA)-PEG5K 1	0.057±0.010	3.9±0.6
	P(CL/GA)-PEG5K 2	0.039±0.003	1.8±0.5
	P(CL/GA)-PEG5K 5	0.046±0.008	2.6±0.6
	Negative control	0.023±0.006	-
	Positive control	0.895±0.025	-

ACKNOWLEDGEMENTS

This work was supported by National Natural Science Foundation of China (No. 81570287), Science and Technology Development Plan of Shandong Province (No. 2015GGX102017 and 2018GGX102016), Achievement Transformation Plan of Qingdao (No. 16-6-2-17-nsh and 16-6-2-9-nsh).