Ariane Pinche

CREMMALab Project

Ariane Pinche École nationale des chartes, Centre Jean Mabillon DH-Tokyo 2022 Within the infrastructure of the CREMMA project (Consortium for Handwriting Recognition of Ancient Materials) supported by the DIM (research funded by the Île-de-France Region) MAP (Ancient and Heritage Materials), the CREMMALab 1 project combines research questions, creation and release of data from French medieval literary manuscripts for HTR (see the cremma-medieval repository on Github : https://github.com/HTR-United/cremma-medieval).

The challenge of HTR is still to be taken up because of the great variety of writings, the poor readability, even for a human reader, of handwritten documents, whether due to the degradation of the source or the lack of homogeneity of the writing. Finally, the production of training data is extremely costly. However, the technical progress of the last few years in AI and neural networks allows us to produce data that significantly reduces manual work. In the Humanities, if some tools have emerged such as Transkribus [START_REF] Kahle | Transkribus-A Service Platform for Transcription, Recognition and Retrieval of Historical Documents[END_REF] or Kraken [START_REF] Kiessling | Kraken-An Universal Text Recognizer for the Humanities[END_REF] and its interface eScriptorium [START_REF] Kiessling | eScriptorium: An Open Source Platform for Historical Document Analysis[END_REF], developed at the EPHE, PSL university, we lack data on medieval documents to train performant models. So, producing training data is today a major issue.

The objective of the CREMMALab project is to propose open training data and HTR models for medieval documents. All data and models produced by the project are already available in the cremma-medieval repository on HTR-united [START_REF] Chagué | HTR-United, a centralization effort of HTR and OCR ground-truth repositories mainly for French languages[END_REF]. The CREMMALab project implements transcription protocols to optimize the training of HTR models and to eventually produce homogeneous and shareable data. As a first step towards the FAIR principles, through the cremma-medieval repository, we have set up some tools to ensure the citability, the durability and the quality of the data. Thus, data are described (language, date, type of document, transcription method, number of transcribed lines etc.). Then thanks to continuous integration tools, the compatibility of the XML data is checked (HTRUX), as well as the uniformity of the character sets used in the corpus (chocoMufin). Through the gathering of a corpus of medieval manuscripts, the learning process of the HTR algorithms is examined to evaluate the problems related to the constitution of training data : how to transcribe, handle abbreviations, segment words and so on. We also seek to determine thresholds of ground truth lines needed to meet quality goals, and also to evaluate the impact of the training corpus on the quality and genericity of the models.

Bicerin, an HTR model for medieval French manuscripts, is already available. It has been trained on eleven manuscripts written between the 13 th and 14 th centuries in Gothic script (about 18400 transcribed lines, see table 1). Its accuracy (CER) on this corpus is 95.38 % (dev score). This generic model, which still needs to be improved produces predictions for similar out-of-domain sources (Chantilly, Bibliothèque du Château, ms. 734, 14 e siècle) with an accuracy of 83 % (test score, see figure 1). A quick customization, requiring the addition of only two folios (336 transcribed lines) achieves an accuracy of 91 % (test score with the same sample as the first experiment, see figure 2). Most of the recognition problems come from specific difficulties due to the manuscript : word segmentation, distinction between u and n, abbreviations and reproduction quality.

The model can also be customized on a document which, at first sight, might seem incompatible with the training corpus. The manuscript codex 909 from the University of Pennsylvania was written in France at the end of the 15 th century, or at the very beginning of the 16th century. It is written in Bâtarde (this script is a hybrid of the formal style with a cursive script). Applied directly, the model shows an accuracy of 80 % (test score, figure image 3). However, Bicerin is very flexible, and with a customization from the addition of two folios (320 manually transcribed lines), we get an accuracy of 97 % (test score with the same sample as the precedent experience, see figure 4). The high recognition of this writing is certainly related to the regularity of the writing and the quality of the support and the reproduction, two criteria that seem to be more important for HTR than the type of writing.

In the future, we hope to increase the number and diversity of our training data to improve the genericity of the model and its robustness. We also hope to determine its breakpoints and to delimit contexts in which a generic model is enough and those in which it is relevant to create a personalization or another model from scratch.

Annexes

Figure 2 :Figure 4 :

 24 Figure 2: Bicerin model prediction on Philadelphy, university of pennsylvania, ms codex 909

Table 1 :

 1 Cremma medieval corpus

	Manuscript	Date	Transcribed Lines
	BnF, ms fr. 17229	13th	161
	BnF, ms fr. 13496	13th	159
	BnF, ms fr. 411	14th	153
	BnF, Arsenal 3516	13th	1991
	BnF, ms fr. 22549	14th	411
	BnF, ms fr. 24428	13th	1295
	BnF, ms fr. 412	13th	4551
	BnF, ms fr. 844	13th	1026
	Cologny, bodmer, 168	13th	1927
	Vaticane, Reg. Lat., 1616	14th	1726

Figure 1: Bicerin model prediction on Chantilly, ms. 734


PHD Vincentrialle

MD,MSC,CSPQ ) Emmanuelstip

PHD ) Kierono'connor

COMPUTER-MEDIATEDPSYCHOTHERAPYETHICALISSUESANDDIFFICULTIESIN IMPLEMENTATION

Computer technology has been applied to psychiatry and psychotherapy for four main purposes: as a diagnostic aid, for computer-assisted instruction, to aid cognitive rehabilitation, and for computer-mediated psychotherapy (CMP). The first and second applications have proved highly useful in some psychiatric domains, but CMP is at a preliminary stage and remains controversial because little evaluation data is available. The questions addressed in this paper are: Can psychologic computer software cope with emotional and behavioural dysfunctions? To what extent can the use of computers help the patient recover? The authors review experiences in this area and discuss the dangers, possible drawbacks, major objections and limits of CMP. They also examine the possibility that computer applications may help in the understanding of psychotherapy. Finally, they address some ethical issues in the application of this technique. More and more physicians use computers in their offices. Interactive computer software has been applied to psychiatry and psychotherapy in 4 main areas: first, to aid diagnosis, for mental-state assessment and as a therapeutic accessory1-4 (for which there are more than 15 tentative decision-making expert systems5,6); second, for evaluating students' knowledge;3,7 third, to aid cognitive rehabilitation;8 and fourth, for computer-mediated psychotherapy (CMP), which has been used to direct changes in patient behaviour.9-18 Although the first three purposes have proved highly useful in specific domains, CMP is at a preliminary stage and remains controversial and poorly evaluated. Thus, the central questions addressed in this paper are: Can psychologic computer software help individuals with emotional and behavioural dysfunctions? To what extent-can they help? What have been the experiences in this domain? and What are the dangers, drawbacks, objections and limits of CMP? Finally, we discuss the ethical issues that must be addressed before large-scale evaluations are undertaken. Evaluating the efficacy of computer use is straightforward when the task outcome goals are easy to assess; however, when computers are used for psychotherapy, we first must determine what stages and processes are involved.

Successful Experiments

Experiments with computers as a medium for psychotherapy have been attempted since the 1960s when Weizenbaum19 designed his ELIZA program. [START_REF] Demuth | Eliza and her offspring[END_REF] The program is one of the most famous attempted realizations of artificial intelligence: a branch of computer science devoted to the study of artificial reasoning, problem-solving, knowledge representation and automatic knowledge learning. Weizenbaum set out to demonstrate that a computerized model of a psychoanalyst would be no more than a pale reflection of the human it pretended to mimic. Much to his surprise, the program became a prototype for CMP and, more than 18 years after its presentation, an updated version of the program was still in use, and positive results are still being reported.21 This initial experiment was followed by a series of positive reports on CMP. Biglan and associates22 successfully treated a volunteer group of 15 test-anxious students by means of behaviourally oriented, computer-administered desensitization. Foree-Gavert and Gavert23 and Burnett, Taylor and Agras24 proposed the computer-assisted selftreatment of obesity. Ghosh and Marks25 devised a computerized behavioural program for the self-exposure treatment of phobias. They compared patients treated by the computer with a group of patients who received conventional treatment by a therapist or through a self-help manual and found that at the end of treatment patients in both groups showed similar, significant improvement in their clinical state. Ford and Vitelli26 surveyed 72 prison inmates who had participated in both computer assessment and a Computer Adjunct to Psychotherapy (CAP) program. Their preferences were almost equally divided between CAP and human-conducted therapy sessions. Reitman27 used microcomputers in self-help sex therapy, and Binik, Servan-Schreiber and coworkers28,7 developed SEXPERT -a rule-based, computer-mediated system for the assessment and treatment of sexual dysfunction in individuals and couples. These last authors29 stressed the ability of their system to seriously engage couples, to encourage them to discuss their sex lives and relationships with each other and to interact with the system's comments. Computers have been used as an aid to prevent drug abuse. Barber29 described two computer-assisted learning programs: one for the prevention of relapse in former heroin users and the other for the promotion of controlled drinking in early-stage problem drinkers. Computer-assisted, short-term psychotherapy is said to be effective in people who have problems in living. In each of five 2-hour sessions the therapist introduces the patient to a concept to be worked on with the computer, and then talks to the patient about the results.30 Kim and Kim31 described the development of an expert system for brief psychotherapy, and Neumann32 used the computer to modify the individual's perceived competence to effectively operate in the everyday environment.

Computers as Life Tools

An important factor in CMP is that the patient attributes certain characteristics to the computer. In clinical settings, subjective assessments after computer use show a high degree of acceptance. Contrary to early fears that computers would have a dehumanizing effect, people view computers as credible sources of information and as reliable tools for decision making.33 This acceptance of computers in psychiatry mirrors their acceptance in everyday life. After their explosive diffusion in the '80s, computers are now commonplace. Devices such as the Minitel (France-Télécom, Paris) and microcomputers connected by a modem to telephone networks give many people access to a huge pool of databases and services. Computer use now begins in primary school in almost all countries where the standard of living permits.

Consequently, the computer' s broad social acceptability should significantly influence the debate about its place in psychotherapy. Clinicians and researchers in psychiatry and psychotherapy have tried to assess patient acceptance of computer programs for mental-state assessment or treatment. [START_REF] Ekstrom | Special issue: Computers in counseling and development[END_REF] Almost none reported a negative experience. Erdman and collaborators35 expressed one aspect.

[We] readily concede that some patients might find a computer terminal a dehumanising medium for dealing with major 4fe issues. But it is equally true that many people find it embarrassing and humiliating to divulge the intimate details of their lives to a stranger, no matter how wrapped in the cloak of professionalism. Users report that their relation with the computer is no more dehumanizing than their relation with the automobile or any other commonly used device. Computers are credible sources of information, even for those who might find interaction slow and difficult, such as people suffering from vertigo33 or obsessional neurosis.36

Computers and Directive Therapies

The most successful applications use standard behavioural packages or open-ended nondirective packages that achieve therapeutic effects mainly by stimulating reflection. Could the computer be effective in a more insight-oriented approach, or through the increasingly popular, thought-modifying techniques of cognitive therapy? What qualities are perceived as necessary for psychoanalytic and cognitive therapists? Psychodynamic therapy is a procession of stages requiring a variety of therapist skills. The educational stage involves evaluating, formulating probable diagnoses and providing patients with information on the source of their difficulties. In the empathicconnection stage the therapist forms a therapeutic alliance with the patient, explores deep conflicts and develops transference-countertransference relations, with subsequent projection or projective identification. To interpret, direct and elaborate psychic processes the therapist requires a variety of eclectic techniques. The last stage is the termination and follow-up.

Human Emotion

Qualities required by a therapist are intelligence, patience, listening and observation skills, understanding, self-control, consideration, affability, sociability, the ability to support, protect and interpret, and the Rogerian triad of warmth, empathy and genuineness. In addition, the therapist may become an idealised object or an authority figure. Clinicians already fear that the drive for cost-efficiency is forcing the human therapist to forego these qualities.37 Computers cannot provide this range of human emotion, so we must determine to what extent these qualities are necessary to induce change. Perhaps they are necessary only when a human is inducing the change? For example, in the presence of another human, the patient may require immediate reassurance that he or she will not be rejected. Also, the patient probably feels bound to project human qualities onto the therapist. However, these projections may be superfluous to actual change in the patient's thought and emotional processes. The main component of human psychotherapy is the relationship between the therapist and the patient, viz. transference and countertransference.

Object-Relations

We might look at the relationship with a computer from a Kleinian "object-relations" point of view: one that sees personal development as the product of the way the self interacts with objects in the immediate environment. In Freudian theory it is important to consider the objects and the significance of the influence, of those objects on internal psychic development. Infants begin life in a state of objectless primary narcissism and progress in the direction of object libido and attachment. As are many other objects (cars, televisions), the computer is a locus for libido investment. Thus, the computer-relations theory is based in specific object-relations. Computers contain a great deal of information, therefore the human sees interaction with them as a relationship between two knowledge systems. Some psychiatric states (e.g., major depressive disorders) are secondary to the way in which individuals think about themselves and their role in the world. For them, knowledge-based interactions could be useful.

Cognitive Therapy

Cognitive therapy is based on the notion that psychiatric disorders are secondary to stereotyped thoughts that lead to cognitive distortions, errors in thinking and selfdefeating behaviour. One of the psychotherapist's tasks is to guide the patient in selecting among possible behaviours and to evoke a kind of "problem-solving" therapy. Success 'is determined by the patient's computational resources. It is adaptive to have stable, pre-existing solutions to thinking errors. Straightforward interaction between patients and problem-solving software could aid their adaptation. Cognitive therapy is becoming increasingly technical in its approach to modifying the patient's verbal behaviour. Caro38 produced a linguistic-based cognitive therapy, centered on sentence construction and phraseology, to evaluate irrational sentences. Cognitive exercises such as relabelling, reality testing, reattribution and rational inference can be carried out with current software. In a rule-based system like the Beck-style cognitive therapy,39 the computer detects and feeds back maladaptative statements.

Anticipated Difficulties

A computer would have difficulty in actively seeking information during evaluation and treatment; for example, without special sensors, a computer could not detect verbal nuances or nonverbal behaviour such as discomfort in the interaction. Computer vocabulary can never reflect the richness of conscious experience. It cannot role-play or give credible reassurance and empathy. However, as mentioned earlier, some human qualities may be complicating rather than cathartic factors. The patient would have fewer emotional attachments with the computer, his or her feelings of mastery would be enhanced and there would be no possibility of exploitation, abuse or boundary violation. Also, because the patient would probably perceive that the computer had a lower status, he or she might find it easier to reveal intimate or compromising information. (The fear of negative evaluation by a high-status professional would be absent.) Slack and associates40 have shown that in the absence of therapists a computer interview can provoke both anxiety and relaxation responses. This was confirmed by significant changes in heart rate and in anxiety-state scores. In the opinion of these authors the therapist's presence can sometimes inhibit frank disclosure. Computers can help using visual-spatial and auditory modes; human therapists generally operate verbally.

We need to address the technical problems of interaction (particularly with elderly people) in terms of manual-operation posture and visual display and, for a minority of users, the danger of computer dependence. [START_REF] Logie | Computers and patient care[END_REF] The development of the computer as a cognitive or interpretative therapist depends equally on increased sophistication and acceptance of computers and on precise analysis of therapy components. Computer connectionist modelling has helped us to form the therapeutic process.42 ("Connectionism" is the study of artificial neural networks of individual processing units with collective properties similar to networks of biologic neurons.) If we can model therapeutic change entirely by information-processing concepts, a computer may replace the traditional psychodynamic stages associated with the human psychotherapist. However, in the near future it is likely that CMP programs will be the most successfully used applications.42

Limitations of Computer Modelling in Psychotherapy

Despite the progress of cognitive science in modelling symbolic knowledge and reasoning, neural networks, the brain and human memory, in our attempts to design a model of a whole therapeutic behaviour we run the risk of adopting a restricted view of the human being. Can one expect the computer to be anything more than a powerful information tool or at best, an information-processing device? On this point, Nadelson43 is sceptical. We, as therapists with all of our all-too-human faults, will always be better than the computer at the care of the patient; we need help in the treatment of the disease. There is no real issue of replacement of human therapists by a machine. We conclude that any CMP application must be sufficiently structured and delimited to be described in a computer-programming language. In the near future we cannot expect computerized, long-term, intensive, insight-oriented psychotherapy. However, we have successful, computerized, psychotherapeutic self-treatments for such conditions as obesity, test anxiety, phobias, cigarette smoking44 and some forms of sexual dysfunction. When extended to limited, rule-based, cognitive domains these can provide a valuable resource for structural procedures such as decision making. Undeniably, the computer can replace the therapist for stages of therapy that require straightforward information handling and feedback.45

Ethical Issues

The concept of "computer therapist" raises fundamental ethical problems. Do we run the risk that a patient might make an unwarranted psychologic investment in the software and lock him or herself into a relationship with a computer? Will it be necessary for the program to know at which moment the relationship must terminate? Indeed, how would a computer terminate a relationship? Without human intervention we cannot use the computer as a means of avoiding personal interaction or as a compensation for interpersonal inadequacy. To prevent the perverse use of psychologic softwares we must not use CMP without the supervision of a human therapist. Free access to computerized psychologic assistance can provide advantages to the health care system, because use of the computer would mean a dramatic decrease in the costs of therapy,35 thereby making it more accessible. Nevertheless, there is a risk of commercial abuse if society does not establish controls. The diffusion of CMP software in the marketplace should be supervised closely by a committee of health professionals. Klepsch36 reported that confidentiality was the major concern among his patients with obsessive disorders who used computers for their therapy. We would be capable of divulging, or opening to public scrutiny or peer review, the information gathered by an artificial intelligence product. The computer would offer no resistance because it acts as a perfectly docile executive. On the other hand, the computer would be of considerable use in research to collect impartial data on a sample of patients without the risk of interview bias.

Conclusions

Research into the development of artificial intelligence and computer-assisted instruction has demonstrated that in certain settings computers may perform as well as humans and books in teaching, reasoning, explaining, demonstrating, questioning and answering. The increased use of computers has made it quite natural for people to strum on the keyboard, click on a mouse, navigate through pop-up menus and "talk" to a computer. This should allay our fears concerning the impersonal and dehumanizing nature of computer interviews. We are now ready to address the questions raised by the arrival of psychologic software in the marketplace. The growing investment in research and the subsequent development of knowledge-engineering tools, tutoring systems, friendly usercomputer interfaces, and increasing facilities to design, build and test interactive computer programs constitute the basic resources of CMP software design. Nevertheless, these programming facilities are only the building blocks of the CMP software; the development of this software for human interactive use will be the work of an interdisciplinary team of psychotherapists, cognitive psychologists and computer scientists. Software designed to help individuals eliminate unwanted behaviour, transcend psychologic difficulties or initiate new behaviours cannot be intrinsically malevolent. Its use should be supervised by psychotherapists for two reasons: first, to ensure matching of the software to the patient; and second, to evaluate its long-term use by the patient. One of the main issues raised by the emergence of software programs for CMP is the necessity for qualitative evaluation of the products. The establishment of objective criteria for evaluation will not be easy. In the same way that some books are more pertinent and useful than others, top-level CMP programs may gain the approval of psychotherapists. If this happens, such acceptance will likely represent the major normative criterion for evaluation. However, if CMP remains a matter of controversy, it may be because "the intrusion of artificial intelligence ... into a field so quintessentially human . . . brings into focus the more general problems of psychotherapy."43 Already, there is an interaction between computers and therapists. They play a part in modelling the therapeutic process, they influence other therapies that are becoming increasingly component-based and technique-oriented. As knowledge increases, convergence of the functions of computer and therapist may promote acceptance of computers in therapy.


Evgeniia Gerasimovich

Svetlana Bozrova

Maria Baryshnikova

Zinaida Sokolova
email: zasokolova@mail.ru

Pavel Samokhvalov
email: p.samokhvalov@gmail.com

Chris Guhrenz

Nikolai Gaponik
email: niko-lai.gaponik@tu-dresden.de

Alexander Karaulov
email: drkaraulov@mail.ru

Igor Nabiev
email: igor.nabiev@univ-reims.fr

Alyona Sukhanova
email: alyona.sukhanova@univ-reims.fr

In Vivo Assessment of Acute Toxicity and Immunotoxicity of Quantum Dots with Different Physicochemical Properties

Keywords: quantum dots, semiconductor nanocrystals, toxicity in vivo, immunotoxicity, cytokine 33 profile Gerasimovich, E

come   L'archive ouverte pluridisciplinaire

Introduction

Quantum dots (QDs) are semiconductor fluorescent nanocrystals with a size from 2 37 to 10 nm [START_REF] Medintz | Quantum Dot Bioconjugates for Imaging, Labelling and Sensing[END_REF]. Their optical characteristics such as broad excitation and narrow emission 38 spectra, high photostability and size-depending fluorescence peak spectral position 39 make them a promising tool in the biomedical field [START_REF] Matea | Quantum Dots in Imaging, Drug Delivery and Sensor Applications[END_REF]. Possible biomedical applications 40 of QDs include their use in bioanalytical systems, cell and tissue imaging, drug delivery 41 and tracking in vivo [START_REF] Bilan | Quantum Dot-Based Nanotools for Bioimaging, Diagnostics, and Drug Delivery[END_REF][START_REF] Brazhnik | Quantum Dot-Based Lab-on-a-Bead System for Multiplexed Detection of Free and Total Prostate-Specific Antigens in Clinical Human Serum Samples[END_REF][START_REF] Wagner | Quantum Dots in Biomedical Applications[END_REF]. However, the potential toxicity of QDs is a serious limitation to A number of studies concerning QDs toxicity clearly demonstrated that they can accumulate in various organs and tissues and cause pathological effects. The liver is shown to be one of the main organs for the accumulation of QDs [START_REF] Lu | Review of Toxicological Effect of Quantum Dots on the Liver[END_REF]. In the study of hepatotoxicity of CdSe/ZnS QDs with carboxyl groups they were shown to cause hepatocytes damage and liver inflammation involving inflammosome activation and increased secretion of IL-1β cytokine [START_REF] Lu | CdSe/ZnS Quantum Dots Induce Hepatocyte Pyroptosis and Liver Inflammation via NLRP3 Inflammasome Activation[END_REF]. Similarly, in the study of Cd/Se/Te-based QDs with ZnS shell the elevation of IL-6 and TNF-α level were shown, although no histopathological damage was observed [START_REF] Lin | Cd/Se/Te-Based Quantum Dot 705 Modulated Redox Homeostasis with Hepatotoxicity in Mice[END_REF]. The study of in vivo biodistribution of CdTe aqueous QDs stated the initial accumulation of QDs in liver and their further absorption in kidney during long-time blood circulation [START_REF] Su | In Vivo Distribution, Pharmacokinetics, and Toxicity of Aqueous Synthesized Cadmium-Containing Quantum Dots[END_REF]. In addition, it was showed that Cd-containing QDs disrupted the redox balance of primary kidney cells, thus inducing their damage [START_REF] Zhao | Kidney Toxicity and Response of Selenium Containing Protein-Glutathione Peroxidase (Gpx3) to CdTe QDs on Different Levels[END_REF]. In conclusion, many evidences demonstrate that QDs can accumulate in major organs and cause pathological damage [START_REF] Liang | Toxicity of Quantum Dots on Target Organs and Immune System[END_REF].

After the intravenous administration QDs were shown to accumulate in the spleen and thymus and maintain fluorescence ability for a long time after injection [START_REF] Fitzpatrick | Long Term Persistence and Spectral Blue Shifting of Quantum Dots in Vivo[END_REF]. The results of immunotoxicity study of CdSe/ZnS QDs show that the majority of administered QDs are taken up by the immune organs (spleen and thymus). Lymphocytes from QD-treated mice exhibited lower viability and increase of the release of TNF-α and IL-6 [START_REF] Wang | Immunotoxicity Assessment of CdSe/ZnS Quantum Dots in Macrophages, Lymphocytes and BALB/c Mice[END_REF]. The study of the acute toxicity of CdSe/CdS-MPA QDs after repeated intraperitoneal injection demonstrated the accumulation of QDs in the liver and spleen and elevated level of IL-6 in these organs and plasma [START_REF] Haque | Acute Toxicity and Tissue Distribution of CdSe/CdS-MPA Quantum Dots after Repeated Intraperitoneal Injection to Mice[END_REF]. The study of intraperitoneal injection of PEG-InP/ZnS QDs in mice showed the increase of the percentage of neutrophils and IL-6 levels in mouse PLF (peritoneal lavage fluid) and plasma, leading to the acute-phase inflammation in mice [START_REF] Chen | InP/ZnS Quantum Dots Cause Inflammatory Response in Macrophages through Endoplasmic Reticulum Stress and Oxidative Stress[END_REF]. Intravenous administration of CdTe QDs resulted in the alteration of the level of several pro-inflammatory cytokines. Specifically, IL-6 level was significantly increased starting at the dose of 0.4 mg/kg, while IL-12 (p70) and TNF-α levels were elevated starting from 5 mg/kg, but showed significant reduction at the highest doses [START_REF] Nguyen | Biodistribution and Systemic Effects in Mice Following Intravenous Administration of Cadmium Telluride Quantum Dot Nanoparticles[END_REF]. In contrast, immunotoxicity study in vivo demonstrated little effect of CdInS2/ZnS QDs on immune organs and the level of pro-inflammatory cytokines [START_REF] Chen | In Vitro and in Vivo Immunotoxicity of PEGylated Cd-Free CuInS2/ZnS Quantum Dots[END_REF].

Another study revealed the dependence of immunotoxicity on molecules used for surface coating of QDs [START_REF] Dai | AMP-Conjugated Quantum Dots: Low Immunotoxicity Both In Vitro and In Vivo[END_REF]. Summing up, the immunotoxicity of QDs can be affected by QD type, surface modifications and dose in a non-linear manner.

Assessment of cytokines concentration is one of the indicative methods for the immunotoxicity studies [START_REF] Corsini | Evaluating Cytokines in Immunotoxicity Testing[END_REF]. The cytokines are divided into two groups: the pro-inflammatory such as IL-6 [START_REF] Tanaka | IL-6 in Inflammation, Immunity, and Disease[END_REF], TNF [START_REF] Zelová | TNF-α Signalling and Inflammation: Interactions between Old Acquaintances[END_REF], IL-12 (p70) [START_REF] Vignali | IL-12 Family Cytokines: Immunological Playmakers[END_REF], IFN-γ [START_REF] Schroder | Interferon-γ: An Overview of Signals, Mechanisms and Functions[END_REF] and anti-inflammatory such as IL-10 [START_REF] Vignali | IL-12 Family Cytokines: Immunological Playmakers[END_REF]. Monocyte chemoattractant protein 1 (MCP-1) is one of the key chemokines which regulates migration and infiltration of monocytes/macrophages during inflammatory response [START_REF] Deshmane | Monocyte Chemoattractant Protein-1 (MCP-1): An Overview[END_REF]. Thus, the alterations in cytokines and chemokines concentration can reflect the reaction of the immune system to the administration of potentially toxic substances.

Our study aimed to assess the acute toxicity in vivo of different types of QDs by calculating the LD50 values for all types of QDs, estimating the physiological responses of laboratory animals on the injection of QD preparations, and toxic effects of QDs on different organs. In addition, we used the most toxic CdSe/ZnS-PEG-OH QDs to investigate the immunotoxicity after intravenous administration in mice. For this purpose, six cytokines (IL-12p70, TNF, IFN-γ, MCP-1, IL-6, IL-10) concentration was measured to assess the potential inflammation response after exposure to CdSe/ZnS-PEG-OH QDs.

Materials and Methods

Methanol, chloroform, sodium phosphate dibasic, sodium phosphate monobasic, sodium hydroxide, DL-cysteine hydrochloride hydrate were purchased from Sigma-Aldrich, US. PEG derivatives HS-(CH2)11-EG6-OH, HS-(CH2)11-EG6-OCH2-COOH, HS-(CH2)11-EG6-NH2 were purchased from ProChimia Surfaces Sp, Poland.

Quantum dots synthesis and solubilization

CdSe/ZnS (core/shell) quantum dots with a fluorescence maximum at 592 nm were synthesized as described earlier [START_REF] Nabiev | Engineering of Ultra-Small Diagnostic Nanoprobes through Oriented Conjugation of Single-Domain Antibodies and Quantum Dots[END_REF]. The synthesis of CuInS2/ZnS and PbS/CdS/ZnS QDs was carried out by the method described in [START_REF] Vokhmintcev | Two-Stage ZnS Shell Coating on the CuInS2 Quantum Dots for Their Effective Solubilization[END_REF] for CuInS2/ZnS QDs. After the synthesis quantum dots were transferred from organic phase to water solution by replacing hydrophobic surface ligands with polyethylene glycol derivatives following the procedure described earlier [START_REF] Nabiev | Engineering of Ultra-Small Diagnostic Nanoprobes through Oriented Conjugation of Single-Domain Antibodies and Quantum Dots[END_REF]. Briefly, quantum dots were dissolved in chloroform and precipitated with 10 mg/ml DL-cysteine solution in methanol. The excess of DL-cysteine was washed off with methanol. The precipitate was dried and then dissolved in a weak alkaline solution. according to the manufacturer's protocol. For this purpose, 500 μL of a QD solution was applied onto a column preliminarily equilibrated with a 0.1 M sodium phosphate buffer solution with the corresponding pH. After that, 1 mL of the same buffer solution was used for elution. The fractions containing QDs were collected into a separate test tube. The purification procedure was performed twice. The resultant solution was sequentially filtered through Millex-GV 0.22 μm and Whatman Anotop 0.1 μm filter units. The solution of PEG derivatives with different functional groups (PEG filtrate solutions) were prepared by centrifugation of QDs solution using Amicon Ultra-4 centrifugal filter units with a 10 kDa cut-off (Millipore).

Quantum dots characterization

For the determination of QDs concentration at the final solution we used a weight method. The aliquot of 35 μL of the final preparation of the QDs solution was placed into a preliminarily weighted 0.5-mL low-bind test tube (Eppendorf) and then dried in the Concentrator Plus (Eppendorf, Germany) for 3 h at the temperature of 30°C. After that, the test tube was weighted again. The quantity of QDs contained in 35 μL of the original QD solution was calculated by subtracting the initial weight of the empty test tube from the final weight of the test tube containing the QD preparation after drying. The QD quantity per milliliter of solution was calculated to obtain the mass concentration. The hydrodynamic diameter and zeta potential of solubilized QD were measured by dynamic laser scattering and laser Doppler electrophoresis, respectively, by using Zetasizer Nano-ZS device (Malvern Instrument Ltd., UK).

Estimation of acute cytotoxicity of QDs in vivo

The analysis of the acute in vivo toxicity of the QD preparations, including the estimation of their median lethal doses (LD50), was performed in experiments on female BALB/c mice obtained from the vivarium of the Blokhin Russian Cancer Research Center of the Ministry of Health of Russia. Standard animals weighing 18-22 g were used in the study. All the animals were healthy; they were kept in special roomy cages at room temperature (20-23°C), a relative humidity of 60-65%, natural illumination, and forced air supply, on a litter of wood shavings sterilized in a hot-air oven. The mice were fed on the standard commercial certified pelletized feed for rodents with a known expiry date.

The mice had free round-the-clock access to pure drinking water. The day when the preparation was injected was taken to by day 0. In that day, the mice were watched for 6 h after the injection. After that, they were examined twice a day for 15 days. The number of the animals that died was the criterion for estimating the acute toxicity of the QDs. The toxicity was measured as the lethal dose (LD), i.e., the amount of QDs causing death of a specified percentage of the animals.

Assessment of CdSe/ZnS-PEG-OH QDs immunotoxicity

Eighty BALB/c mice were treated with 25 mg/kg CdSe/ZnS-PEG QDs. A solvent solution (0.05 M Na-phosphate buffer, pH 7.2) and PEG filtrate solution with the same concentration of PEG as in the CdSe/ZnS-PEG solution were used as negative controls. At the time points of 6, 24, 48 hours and 7 days intact animals was used as an additional negative control. All solutions were injected intravenously in the tail vein. Serum samples were collected at 6, 24, 48 hours, 7 and 30 days after injection. Serum from all mice in one group were pooled to obtain necessacary amount of material and stored at -20°С until analysis.

Mouse cytokines concentration measurement

The concentration of six pro-inflammatory cytokynes was measured using cytometric bead array kit (BD™ CBA Mouse Inflammation Kit, BD Biosciences, California, USA) according to manufacturer's instrusctions. This technique is based on six bead populations with distinct fluorescence intensities coated with antibodies specific to IL-12p70, TNF, IFN-γ, MCP-1, IL-6, IL-10. The cytokine capture beads and phycoerythrin-conjugated detection antibodies were incubated with serum samples or standards to form sandwich complexes. Then samples were measured on BD FACS Canto™ II cytometer (BD Biosciences, California, USA) and FCAP Array™ 3.0 software (BD Biosciences, California, USA) was used to calculate the concentration of cytokines using fluorescence intensity data. The standard curve range for each analyte was from 20 to 5000 pg/ml.

Results

QD synthesis, solubilization and characterization

In the presented study, QDs with various chemical composition and surface charge were used to investigate their toxic effects in mice model. After the synthesis of QDs in the organic phase, we modified their surface with thiol-containing PEG derivatives to obtain aqueous QD preparations. Using mixtures of PEG derivatives with different functional groups for the modification of CdSe/ZnS QDs, we obtained QD solutions with different surface charge. The surface charge and hydrodynamic diameter of QDs were determined using a Zetasizer Nano ZS instrument by the electrophoretic mobility method employing the Doppler effect and by the dynamic light scattering method, respectively. Table 1 shows the sizes and charges of the QDs used in the study.

Acute in vivo cytotoxicity of quantum dots

The mice were injected with the QDs preparations into the caudal vein, after which we watched the animals carefully for 24 h. After that, the number of surviving animals was recorded.

We estimated the in vivo acute toxicity of QDs with different chemical compositions of the core (Figure 1) and with different surface charges (Figure 2). The data obtained in this experiment were used to calculate the LD50 values for all types of QDs studied (Table 2). Our results showed that CuInS2/ZnS QDs had less toxic effect on mice than PbS/CdS/ZnS and CdSe/ZnS QDs, despite their surface charge were relatively similar due to the same PEG derivative used for their modification (Fig. 1). Furthermore, the obtained means of LD50 for QDs with the same chemical composition of the core (CdSe/ZnS) but carried different surface charge varied considerably. CdSe/ZnS-PEG-OH QDS with low negative charge exhibited the highest toxic effect on mice, while the LD50 of CdSe/ZnS-PEG-NH2 QDS with low positive charge turned out to be approximately two times higher. CdSe/ZnS-PEG-COOH QDs were showed to cause the weakest toxic effect on mice. 3. The data show that 24 hours after administration of CdSe/ZnS-PEG QDs the level of IL-12 was slightly elevated compared to control group and groups treated by buffer and PEG solutions, but starting from 48 hours the level of this cytokine was low in all groups of animals. The concentration of another pro-inflammatory cytokine, TNF, is raised in group treated with CdSe/ZnS-PEG QDs after 48 hours after injection. In addition, the level of IL-6 was somewhat higher 6 hours after injection in the groups treated with buffer, PEG and CdSe/ZnS-PEG QDs solutions compared to intact animals, but after longer time periods it remained at the basic level. The level of anti-inflammatory cytokine, IL-10, was below the limit of detection in samples from all experimental groups. The level of MCP-1, pro-inflammatory chemokine, was elevated in groups treated with buffer and CdSe/ZnS-PEG QDs 6 hours after injection. However, 30 days after the start of the experiment the level of MCP-1 was twice higher in groups treated with PEG and CdSe/ZnS-PEG in comparison with group treated with buffer.

Discussion

Semiconductor nanocrystals, or quantum dots, have a great potential in the field of biomedicine, but their use is restricted by potential toxic effects on living organisms. The in vivo toxicity of CdSe/ZnS QDs has been reported in several studies. However, a more systematic evaluation of negative effects of QDs on living organisms is necessary for safe human use. One of the important aspects is an immunotoxicity as the immune system functions include protection from foreign substances.

We have estimated the LD50 values for different types of QDs, physiological responses of laboratory animals on the injection of QD preparations, and toxic effects of QDs on different organs. Such experiments could help to improve the safety testing standards for nanotechnological products based on nanocrystals.

The QD preparations were injected into the caudal vein of BALB/c mice, after which we carefully watched the mice for 24 h and then examined them twice a day for 15 days.

We estimated the in vivo toxicity of QDs with different chemical compositions of the core (Fig. 1) and those with different surface charges (Fig. 2). The results were used to calculate the LD50 values for all types of QDs studied (Table 2). Our data showed that the CdSe/ZnS-PEG-OH QDs with a low negative surface charge were the most toxic. The QDs with a high negative surface charge were the least toxic. It was also demonstrated that the QDs with CuInS2 cores were less toxic than the QDs whose cores contained heavy metals.

In addition, our experiments on toxicity showed that intravenous injections of QDs with different chemical compositions provoked different physiological responses. The administration of CdSe/ZnS and PbS/ZnS QDs caused an increase in the respiration and heart rates, paresis of the legs, convulsive retching movements, and spasm of the caudal vein. After several hours, the surviving animals completely recovered. After the injection of CuInS2/ZnS QDs, the mice felt well for 5-10 min, after which leg paresis, rapid and shallow breathing and general paralysis were observed. These transitory disorders suggest the formation of metastable QD aggregations in the blood, which either were broken up due to opsonization and absorbed without affecting considerably the general state of health or, in more acute situations, led to death of the mice. There are published data that QDs may cause lung thrombosis [START_REF] Geys | Acute Toxicity and Prothrombotic Effects of Quantum Dots: Impact of Surface Charge[END_REF], which explains the condition of the mice in our study. The mechanism of QD-induced thrombosis is unknown; however, it is assumed [START_REF] Geys | Acute Toxicity and Prothrombotic Effects of Quantum Dots: Impact of Surface Charge[END_REF] that QDs are capable of triggering the clotting process in blood. A different physiological response to CuInS2/ZnS QDs suggests that the toxic effect of these QDs is underlain by a different mechanism, apparently determined by copper, which, unlike lead and cadmium, is not a heavy metal [START_REF] Pons | Cadmium-Free CuInS2/ZnS Quantum Dots for Sentinel Lymph Node Imaging with Reduced Toxicity[END_REF].

Our results clearly demonstrate that the QDs surface charge also influences their toxicity. Indeed, the QDs with a low negative surface charge have proved to be the most toxic, and those with a higher negative charge, the least toxic. The observed effect is most likely to be determined by the differences in the distribution and accumulation of QDs in the mouse body and the differences in the interactions with the components of biological fluids. For example, negatively charged QDs may rapidly accumulate in lymph nodes [START_REF] Robe | Quantum Dots in Axillary Lymph Node Mapping: Biodistribution Study in Healthy Mice[END_REF], and electrically neutral QDs are less prone to electrostatic interaction with proteins, which interferes with the formation of the "protein corona" and promotes their degradation [START_REF] Liu | Compact Cystein-Coated CdSe(ZnCdS) QDs for in Vivo Applications[END_REF].

After the most toxic type of QDs was revealed, we performed an additional experiment in order to assess the effect of these QDs on the immune system. For this purpose, we estimated the effect of CdSe/ZnS-PEG-OH QDs at the dose of 25 mg/kg, or LD10, on cytokine profile in mice. Buffer solution (0.1 M Na-phosphate, pH 7.2) and PEG-OH solution were used as controls in the study. The concentration of IL-12p70, TNF, IFN-γ, MCP-1, IL-10, and IL-6 was determined using cytometric bead array kit (Table 3). The results demonstrated a slight increase in the production of two pro-inflammatory cytokines, IL-12 (p70) and TNF, 24 and 48 hours after injection, respectively. The level of

  At the next stage, DL-cysteine ligands were replaced with PEG derivatives with hydroxyl group. We used three types of PEG derivatives: HS-(CH2)11-EG6-OH, HS-(CH2)11-EG6-OCH2-COOH, and HS-(CH2)11-EG6-NH2. For the modification of CdSe/ZnS QDs mixtures of PEG derivatives were used at the following ratios: 70% of HS-(CH2)11-EG6-OH/30% of HS-(CH2)11-EG6-OCH2-COOH, 70% of HS-(CH2)11-EG6-OH/ 30% of HS-(CH2)11-EG6-NH2, and 100% of HS-(CH2)11-EG6-OH. The specified amounts of ligands were added to working mixtures with the corresponding pH values: 0.1 M sodium phosphate buffer solution (pH 7.2) if the HS-(CH2)11-EG6-OH ligand was used, 0.1 M sodium phosphate buffer solution (pH 8.0) in the case of the mixture of 70% of HS-(CH2)11-EG6-OH / 30% of HS-(CH2)11-EG6-OCH2-COOH, and 0.1 M sodium phosphate buffer solution (pH 6.6) in the case of 70% of HS-(CH2)11-EG6-OH/30% of HS-(CH2)11-EG6-NH2. After that, the mixtures were incubated at a temperature of 4°C overnight. After the incubation, the samples were purified from unbound PEG derivatives using Amicon Ultra-15 centrifugal filter units with a 10 kDa cut-off (Millipore) by centrifugation following the adding 15 mL of 0.1 M sodium phosphate buffer (pH 7.2) in the case of HS-(CH2)11-EG6-OH, 0.1 M sodium phosphate buffer (pH 8.0) in the case of the mixture of 70% of HS-(CH2)11-EG6-OH 30% of HS-(CH2)11-EG6-OCH2-COOH, and 0.1 M sodium phosphate buffer solution (pH 6.6) in the case of 70% of HS-(CH2)11-EG6-OH/30% of HS-(CH2)11-EG6-NH2. The centrifugation was performed three times at the room temperature at 4000 rpm for 10 min. Then, the obtained QDs preparations were additionally purified by gel exclusion chromatography on PD MiniTrap 25G columns (GE Healthcare)

  The mice were divided into five groups (four mice each). CuInS2/ZnS, PbS/CdS/ZnS, and CdSe/ZnS QDs modified with the thiol-containing HS-(CH2)11-EG6-OH ligand, as well as CdSe/ZnS QDs modified with the mixture of 70% of HS-(CH2)11-EG6-OH / 30% of HS-(CH2)11-EG6-OCH2-COOH, the mixture of 70% of HS-(CH2)11-EG6-OH / 30% of HS-(CH2)11-EG6-NH2, or 100% HS-(CH2)11-EG6-OH, were administered at doses from 100 to 300 mg/kg as a single injection into the caudal vein. The substances studied were dissolved in sterile 0.1 M sodium phosphate buffer solution (pH 7.2). Sterile 0.1 M sodium phosphate buffer solution (pH 7.2) and sterile filtered solutions of the corresponding modifying ligands in 0.1 M sodium phosphate buffer solution (pH 7.2) served as control solutions.

Figure 1 .

 1 Figure 1. Survival rate of BALB/c mice as dependent on the quantum dot concentration and core composition.

Figure 2 .Table 2 . 2

 222 Figure 2. Survival rate of BALB/c mice as dependent on the quantum dot concentration and charge.Table 2. LD50 values of different quantum dots for BALB/c mice. QD type LD50, mg/kg CdSe/ZnS-PEG-OH 112.5 CuInS2/ZnS-PEG-OH 200 PbS/CdS/ZnS-PEG-OH 150 CdSe/ZnS-PEG-COOH >300 CdSe/ZnS-PEG-NH2 240

Table 1 .

 1 Size and surface charge of the quantum dots used in the study.

	QD composition	Size, nm	ζ-potential, mV
	PbS/CdS/ZnS-PEG-OH 1	32.04±0.87	-10.60±2.92
	CuInS2/ZnS-PEG-OH	16.08±0.51	-6.12±1.81
	CdSe/ZnS-PEG-OH	16.74±0.28	-4.72±0.38
	CdSe/ZnS-PEG-COOH 2	15.37±0.14	-17.80±3.01
	CdSe/ZnS-PEG-NH2 3	22.77±0.36	6.43±1.12

1 PEG-OH denotes 100% of HS-(CH2)11-EG6-OH 2 PEG-COOH denotes a mixture of 70% of HS-(CH2)11-EG6-OH and 30% of HS-(CH2)11-EG6-OCH2-COOH 3 PEG-NH2 denotes a mixture of 70% of HS-(CH2)11-EG6-OH and 30% of HS-(CH2)11-EG6-NH2

Table 3 .

 3 Concentration of cytokines in mouse serum after the injection of CdSe/ZnS QDs.

	Cytokine concentration, pg/ml	Time	control	Na-phosphate buffer	PEG solution CdSe/ZnS-PEG
		6 h	10.26±2.75	10.36±7.06	7.38±7.81	0.00±0.00
		24 h	3.56±0.30	2.37±0.74	0.00±0.00	10.27±1.25
	IL-12p70	48 h	1.50±0.50	0.00±0.00	0.00±0.00	1.85±0.00
		7 d	0.00±0.00	0.00±0.00	0.00±0.00	0.00±0.00
		30 d	N/A	0.00±0.00	0.00±0.00	0.00±0,00
		6 h	21.71±0.74	25.39±10.42	24.51±9.18	17.14±5.23
		24 h	24.69±4.47	18.19±6.72	26.09±0.99	24.87±1.24
	TNF	48 h	26.27±7.69	18.91±0.25	21.18±3.97	36.62±0.99
		7 d	5.25±2.29	4.72±0.90	4.66±0.33	5.70±0.66
		30 d	N/A	2.49±3.22	5.41±0.25	4.84±1.38
		6 h	2.68±0.57	3.12±0.51	3.55±1.34	2.84±0.34
		24 h	2.01±0.48	3.00±0.34	2.56±0.06	2.26±0.83
	IFN-γ	48 h	3.59±1.4	2.84±0.57	2.88±0.40	2.05±0.54
		7 d	0.59±0.00	0.31±0.21	0.00±0.00	1.11±0.11
		30 d	N/A	0.67±0.06	0.81±0.00	0.42±0.13
		6 h	32.06±5.21	79.47±12.2	49.05±5.71	77.72±5.59
		24 h	35.54±0.28	28.98±3.25	20.08±0.00	32.49±3.46
	MCP-1	48 h	20.62±7.36	26.03±1.51	24.73±2.76	21.88±1.26
		7 d	11.44±3.02	13.79±1.22	17.13±2.91	14.48±4.08
		30 d	N/A	7.51±3.52	13.68±0.16	12.7±0.93
		6 h	0.00±0,00	0.00±0,00	0.00±0,00	0.00±0,00
		24 h	0.00±0,00	0.00±0,00	0.00±0,00	0.00±0,00
	IL-10	48 h	0.00±0,00	0.00±0,00	0.00±0,00	0.00±0,00
		7 d	0.00±0,00	0.00±0,00	0.00±0,00	0.00±0,00
		30 d	N/A	0.00±0.00	0.00±0.00	0.00±0.00
		6 h	1.54±0.13	6.20±2.83	3.68±0.74	8.56±1.85
		24 h	1.51±2.13	2.27±0.65	0.00±0.00	0.00±0.00
	IL-6	48 h	0.00±0.00	0.00±0.00	0.00±0.00	1.33±0.43
		7 d	0.62±0.69	0.00±0.00	0.00±0.00	0.78±0.42
		30 d	N/A	0.33±0.47	0.00±0.00	0.00±0.00

QDs solution 6 hours after administration, but after 30 days it was relatively higher in groups treated with PEG solution and CdSe/ZnS-PEG QDs solution. Our results might evidence that injection of CdSe/ZnS-PEG QDs can cause short-term inflammatory reaction in mice. However, after 7 and 30 days after injection of QDs the level of the majority of pro-inflammatory cytokines was similar to that of control groups. Based on these results, we can suggest that immune reaction to CdSe/ZnS QDs at the dose of 25 mg/kg is reversible, but this issue needs further investigation.

Acknowledgments: A.S. and I.N. acknowledge the support of the French Ministry of Higher Education, Research and Innovation and the University of Reims Champagne-Ardenne. We thank Vladimir Ushakov for proofreading the manuscript.

Funding: This work was supported by the Ministry of Science and Higher Education of the Russian Federation through the grant No. 075-15-2021-935 of the ERA.NET-RUS+ project NanoToBio in its part related to the synthesis of nanomaterials. This work was also supported by the French National Research Agency (ANR-20-CE19-009-02) and co-financed by the European Union via the European Regional Development Fund (FreeBioWave project) in its part related to the development of new surface chemistry approaches.

Data Availability Statement:

The data that support the findings of this study are available from the corresponding authors, A.S. or I.N., upon reasonable request.

Conflicts of Interest:

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.