Raphaëlle Maizeray
email: maizerayraphaelle@gmail.com

Delphine Wagner

François Lefebvre

Hélène Lévy- Bénichou

Yves Bolender

Systematic review and meta-analysis Is there any difference between conventional, passive and active self-ligating brackets? A systematic review and network metaanalysis

Keywords: Orthodontic brackets, Self-ligating brackets, Orthodontic appliance design, Treatment outcome, Systematic review, Network meta-analysis

Aim > To test the null hypothesis that there was no difference in treatment efficiency between therapies undertaken with conventional (CBs), passive self-ligating (PSLBs) or active selfligating (ASLBs) brackets. Materials and methods > An electronic search was performed in 3 data bases (Pubmed, Web of Science, Cochrane Library) from their origin up to January 2019. Additional references were hand searched. Search was strictly restricted to randomized controlled trials (RCTs) and split-mouth design studies (SMDs). RCTs and SMDs were initially processed separately and subsequently combined in a network meta-analysis. The following variables were evaluated: treatment duration, number of visits, occlusal outcomes, alignment rate, transverse arch dimensional changes, incisor position modification, rate of space closure, anchorage loss, bond failure, root resorption, perception of discomfort during the initial phase of alignment, time to ligate in or to untie an archwire, periodontal variables, quality of life. Results > On 229 papers, 30 RCTs and 9 SMDs were finally included in this study. Out of 85 comparisons, 16 only revealed statistically significant differences. It was quicker to untie and ligate an 0.014 NiTi arch from/in 6 ASLBs anterior ceramic brackets compared to 6 ceramic CBs. It was also more painful to insert and remove an 0.019×0.025 SS wire in/from PSLB's brackets compared to CB's attachments. Compared to conventional brackets, there was less maxillary incisor proclination with PSLBs in non-extraction cases. Moreover, there was less bleeding on probing 4-5 weeks after bonding with PSLBs compared to CBs brackets. The only significant difference between ASLBs and PSLBs was that alignment was 10 days faster with active self-ligating braces compared with passive self-ligating braces even if treatment duration between ASLBs and PSLBs was not significantly different. The network metaanalysis revealed that IMPA was greater in extractions cases with CBs compared with both ASLBs (+2,5°) and PSLBs (+1,6°). Conclusions > The vast majority of the studied variables did not show any significant differences between the three types of brackets. The most significant findings were that it was quicker to insert and remove archwires from ASLBs compared to CBs, and it was more painful to insert and remove an 0.019×0.025" stainless steel wire in/from PSLBs compared to CBs. The major difference between ASLBs and PSLBs was that alignment was 10 days faster with active self-ligating braces compared with passive self-ligating braces even if treatment duration between ASLBs and PSLBs was not significantly different. Most of the claims put forward by the suppliers were not substantiated by our network metaanalysis.

Introduction

If edgewise brackets were initially introduced by E.H. Angle in 1925, the very first ligaturefree system was developed ten years later in Brooklyn by Stolzenberg [START_REF] Stolzenberg | The Russell attachment and its improved advantages[END_REF]. Self-ligating brackets were later introduced on an industrial scale in the 80's and started then to be used worldwide. These brackets are being categorized in two different types according to the way the archwire is maintained within the bracket slot: active and passive self-ligating brackets. Active self-ligating brackets are exerting an active force through a spring clip onto the archwire in order to maintain it in the slot, whereas passive self-ligating brackets present an additional slide that once closed does not affect the slot lumen, nor exert an active force on the wire. Two pioneers marked the first steps of these brackets: Hanson started to commercialize his active SPEED™ self-ligating bracket from Canada in 1980 [START_REF] Hanson | The SPEED system: A report on the development of a new edgewise appliance[END_REF] whereas Damon introduced his passive SL bracket in the US in the mid-1990's [START_REF] Damon | Treatment of the face with biocompatible orthodontics[END_REF]. According to orthodontic distributors, self-ligating brackets present many advantages: reduced friction, faster archwire changes, full archwire engagement in the slot, increased patient comfort and hygiene, reduced number of emergencies, reduced root resorption, reduced need of extractions due to arch expansion, faster treatments, reduced number of appointments, better results, higher efficiency. According to the latest JCO study [START_REF] Keim | 2020 study of orthodontic diagnosis and treatment procedures : part 1 : results and trends[END_REF], there seems to be a decreased interest in self-ligating brackets in the USA since 53% of American orthodontists have been using self-ligating brackets in 2020, compared to 63% in 2014. The equivalent figure in France was 37% in 2017 [START_REF] Balteau | Diagnosis and treatment procedures in French orthodontic practices[END_REF]. Authors have been reviewing the efficacy of self-ligating brackets with the help of metaanalyses starting with Chen et al. in 2010 [START_REF] Chen | Systematic review of self-ligating brackets[END_REF][START_REF] Fleming | Self-Ligating Brackets in Orthodontics[END_REF][8][START_REF] Papageorgiou | Clinical effects of pre-adjusted edgewise orthodontic brackets: a systematic review and meta-analysis[END_REF][START_REF] Zhou | Canine retraction and anchorage loss self-ligating versus conventional brackets: a systematic review and meta-analysis[END_REF][START_REF] Arnold | The effect of bracket ligation on the periodontal status of adolescents undergoing orthodontic treatment. A systematic review and meta-analysis[END_REF][START_REF] Yi | Root resorption during orthodontic treatment with self-ligating or conventional brackets: a systematic review and meta-analysis[END_REF][START_REF] Yang | Differences between active and passive self-ligating brackets for orthodontic treatment: systematic review and meta-analysis based on randomized clinical trials[END_REF][START_REF] Yang | Effects of self-ligating brackets on oral hygiene and discomfort: a systematic review and meta-analysis of randomized controlled clinical trials[END_REF][START_REF] Yang | Transversal changes, space closure, and efficiency of conventional and self-ligating appliances : A quantitative systematic review[END_REF][START_REF] Malik | Comparison of anchorage loss between conventional and self-ligating brackets during canine retraction -A systematic review and meta-analysis[END_REF]. A recent meta-analysis [START_REF] Wagner | Are self-ligating brackets more efficient than conventional brackets ? A meta-analysis of randomized controlled and split-mouth trials[END_REF] compared conventional and self-ligating brackets without making a distinction between active and passive self-ligating brackets. Authors concluded that the main difference between these two types of braces was that it is faster to untie and ligate selfligated brackets compared with conventional brackets. Other variables such as time of treatment, number of appointments, alignment efficiency, closure of extraction spaces, changes in transversal dimensions, periodontal health, root resorption or occlusal results did not reveal any statistical difference. A meta-analysis comparing in 2017 active and passive self-ligating brackets [START_REF] Yang | Differences between active and passive self-ligating brackets for orthodontic treatment: systematic review and meta-analysis based on randomized clinical trials[END_REF] found that active self-ligating brackets appeared more efficient for alignment. Comparison of each of these two self-ligating systems with conventional brackets has not been done to this date. It is therefore the aim of this network meta-analysis to compare directly and indirectly the following three types of brackets: conventional brackets (CBs), passive self-ligating brackets (PSLBs), and active self-ligating brackets (ASLBs).

Aim

The goal of this study was to identify Randomized Clinical Trials (RCTs) and Split-Mouth Design studies (SMDs) that compared ASLBs, PSLBs and CBs and to investigate clinical parameters in order to highlight a difference in treatment efficiency. The null hypothesis of this network meta-analysis was that there was no difference in treatment efficiency between the three types of brackets: active self-ligating, passive self-ligating or conventional braces.

Material and Methods

Study design: This study is a network meta-analysis.

Material

Search was strictly restricted to randomized controlled trials (RCTs) and split-mouth design studies (SMDs). We carried out a search protocol according to the PICO criteria: Patients: no age restriction, undergoing fixed orthodontic treatment with either active selfligating, passive self-ligating or conventional brackets, with at least one comparison between two bracket types. Intervention: patients undergoing treatment with self-ligating brackets (either active or passive)

Comparison: patients undergoing treatment with an alternative type of self-ligating bracket or with conventional brackets. Outcomes: treatment duration, number of visits, occlusal outcomes such as the PAR and ICON indices, alignment rate, transverse arch dimensional changes, incisor position modification, rate of space closure, loss of anchorage, bracket failure rate, root resorption, pain perception at 4h, 24h, 3 days and 7 days after bonding, pain experience during wire insertion or removal, time to ligate in or to untie an archwire in/from brackets, periodontal variables such as Visible Plaque Index, Gingival Bleeding Index, Clinical Attachment Level, Bleeding on Probing , quality of life. Exclusion criteria were: absence of control group, in-vitro studies, lingual appliances, patients with cleft lip and/or cleft palate, syndromic patients, lack of interest, absence of randomization, lack of statistical data. An electronic search was performed in 3 data bases (Pubmed, Web of Science, Cochrane Library) from their origin up to January 2019. Keywords used were "orthodontic appliances" [MeSH], "brace" [MeSH], "self-ligat". "randomized controlled trial" [MeSH] or "split-mouth design" [MeSH] were added depending on the type of article targeted. Additional references were hand searched in the following three major orthodontic journals from January 2006 to January 2019: the Angle Orthodontist, the American Journal of Orthodontics and Dentofacial Orthopedics, the European Journal of Orthodontics. We screened the bibliography of the selected papers, and the table of contents of these three journals.

Methods

Following data were collected:

year of publication and country of origin details concerning participants (number of participants, intervention and control groups, methods of assessment)

measured outcomes. Two reviewers (DW and RM) who were not blinded to the authors nor the research results, conducted the selection process. They independently reviewed the titles and abstracts of potential studies. Then, the full texts of potentially eligible studies were obtained for further consideration about quality of the study. Consensus was achieved through discussion. Six key methodological criteria were assessed according to the Cochrane Handbook for Systematic Reviews of Interventions (5.1.0) [18]: sample size calculation, random sequence generation, allocation concealment, complete outcome and description of the participants flux (attrition bias), reporting of withdrawals, use of intention to treat analysis. The risk of bias (high, medium, low) was assessed for each included trial. RCTs and SMDs were initially processed separately and subsequently combined in the metaanalysis, according to Lesaffre et al. [START_REF] Lesaffre | The design and analysis of split-mouth studies : what statisticians and clinicians should know[END_REF]. Clinical heterogeneity was assessed for each variable with I2 statistics. When the p-value ≥ .05, a fixed effects model was used. When the p-value < .05, a random effects model was alternatively selected. Methodological heterogeneity and consistency were checked with Q statistics [START_REF] Cochran | The combination of estimates from different experiments[END_REF]. All quantitative data were processed with Review Manager 5.3 (Nordic Cochrane Centre at Cochrane Collaboration, Copenhagen, Denmark, June 2014). We have been comparing two by two the three different types of brackets for each variable when the corresponding data were available. However as shown on figure 1, when two direct comparisons were available (represented by the two first forest-plots) and the third one was missing (between PSLBs and ASLBs), we have been running a network meta-analysis that allowed us to indirectly compare PSLBs and ASLBs (computer software R VERSION 3.4.3, R Project for Statistical Computing). In the figure 2, the first line corresponds to this very comparison between PSLBs and ASLBs and more precisely to its 95% confidence interval. Since this interval comprises the 0 value, the conclusion is that there is no statistically significant difference in treatment duration between ASLB's and CB's. The GRADE quality of each comparison was also evaluated according to four categories (high, moderate, low, very low quality) [START_REF] Guyatt | GRADE guidelines: 1. Introduction-GRADE evidence profiles and summary of findings tables[END_REF].

Results

Study selection

The electronic search generated 226 articles; 3 more studies were added via hand search. Finally, we included the following 30 RCTs and 9 SMDs [52-60] in our network meta-analysis .The participants' average age was 14.7 years. No SMD comparing ASLBs and PSLBs could be found. Characteristics of included studies are represented in table I.

Risk of bias

The majority of the included studies were considered as presenting a low risk of bias ( 23RCTs and 2 SMDs). 10 included studies presented a moderate risk of bias (8 RCTs [START_REF] Pandis | Maxillary incisor torque with conventional and self-ligating brackets: a prospective clinical trial[END_REF][START_REF] Pandis | Self-ligating vs conventional brackets in the treatment of mandibular crowding: a prospective clinical trial of treatment duration and dental effects[END_REF][START_REF] Miles | Porcelain brackets during initial alignment: are self-ligating cosmetic brackets more efficient?[END_REF][START_REF] Pandis | Comparative assessment of conventional and self-ligating appliances on the effect of mandibular intermolar distance in adolescent nonextraction patients: A single-center randomized controlled trial[END_REF][START_REF] Cattaneo | Labio-lingual root control of lower anterior teeth and canines obtained by active and passive self-ligating brackets[END_REF][START_REF] Folco | Gingival response in orthodontic patients: Comparative study between self-ligating and conventional brackets[END_REF]47,[START_REF] Mde | Alterations in plaque accumulation and gingival inflammation promoted by treatment with self-ligating and conventional orthodontic brackets[END_REF] and 2 SMDs [START_REF] Burrow | Canine retraction rate with self-ligating brackets vs conventional edgewise brackets[END_REF]55]). 4 studies presented a high risk of bias (1 RCT [START_REF] Atik | Evaluation of maxillary arch dimensional and inclination changes with self-ligating and conventional brackets using broad archwires[END_REF] and 3 SMDs [52, [START_REF] Pandis | Failure rate of self-ligating and edgewise brackets bonded with conventional acid etching and a self-etching primer: a prospective in vivo study[END_REF]56]). In fact, these 3 SMDs were not randomized. High GRADE quality direct comparisons were available when comparing: -passive and conventional brackets (duration of treatment, number of visits, transversal modifications, IMPA change in non-extraction cases, pain perception during archwire insertion or removal) -active and conventional brackets (changes of maxillary inter-premolar distance, pain perception after bonding, time to insert and remove an archwire in/from brackets).

Results of network meta-analysis

39 of the 229 studies identified in our research were included in our systematic review and network meta-analysis (figure 3). The analysis of methodologic bias is detailed in table II. Out of the 85 comparisons, 16 only revealed statistically significant differences. The most significant findings were that it was definitely quicker to insert and remove an 0.014 NiTi archwire in/from six 0.018 inch anterior ceramic ASLBs brackets from (21.6 seconds with or without assistance) compared to ceramic CBs (122.7 seconds with assistance, 151.8 seconds without assistance) (figure 4ab) [START_REF] Miles | Porcelain brackets during initial alignment: are self-ligating cosmetic brackets more efficient?[END_REF]. It was however more painful to insert and remove an 0.019×0.025 stainless steel wire in/from PSLBs compared to CBs (33.6/100 pain experience during insertion with PSLBs versus 15.5/100 pain experience with CBs; 38/100 pain experience with PSLBs during removal versus 19.7/100 pain experience with CBs) (figure 5ab) [START_REF] Fleming | Efficiency of mandibular arch alignment with 2 preadjusted edgewise appliances[END_REF]. Compared to CBs, there was less maxillary incisor proclination with PSLBs in non-extraction cases (-1.26°) (figure 6). Moreover, there was less bleeding on probing (-0.10) 4-5 weeks after bonding with PSLBs compared to CBs brackets (figure 7). Alignment was as fast when comparing on one hand PSLBs to CBs (figure 8a) and ASLBs and CBs on the other hand (figure 8b). One major difference between ASLBs and PSLBs was that alignment was 10 days faster with active self-ligating braces compared with passive selfligating braces (figure 8c).

Another significant finding was that IMPA change was greater with CBs in cases of lower first premolar extraction compared with PSLBs (+1.6°) (figure 9a) and with ASLBs (+2.6°) (figure 9b). IMPA change was slightly greater with PSLBs compared to ASLBs in these same extraction cases (0.9°) (figure 9c). Inter-molar and intercanine mandibular and maxillary widths did not exhibit any significant modification between the 3 bracket types with two exceptions. Mandibular inter-molar width was increased to a greater extent with PSLBs compared to CBs (figure 10a) whereas mandibular inter-canine width was majored to a greater extent with CBs compared to PSLBs (figure 10b). These differences amounted however to 0.5mm on average. These overall 16 statistically significant differences were balanced by the fact that the 69 remaining comparisons between the three types of brackets did not reveal any statistically significant difference regarding treatment duration (figures 1,2), number of visits, occlusal outcomes, rate of space closure, anchorage loss, bond failure, root resorption and quality of life.

Discussion

Network meta-analysis, also called multiple treatments meta-analysis, is a popular way to combine evidence on multiple studies (usually randomized trials) comparing multiple treatments [START_REF] White | Network meta-analysis[END_REF]. Its key feature is the ability to combine direct and indirect evidence; for example, the comparison of treatments A and B is performed both using studies that directly compare A with B (direct evidence) and using studies that compare A with C and B with C (indirect evidence). Factors such as the total number of trials in a network, number of trials with more than two comparison arms, heterogeneity (clinical, methodological, and statistical variability within direct and indirect comparisons), inconsistency (discrepancy between direct and indirect comparisons), and bias may influence effect estimates obtained from network meta-analyses [START_REF] Li | Methods Meeting Working Group. Network meta-analysis-highly attractive but more methodological research is needed[END_REF]. Heterogeneity was here evaluated through Q and I ² statistics whereas consistency was checked with a Cochran's Q test. Bias was graded according to the Cochrane Handbook for Systematic Reviews of Interventions. The most clinically interesting differences between ASLB's and CB's were the extremely reduced time to tie or unligate an archwire. This difference explains by itself the popularity encountered among health professionals by these self-ligating brackets [START_REF] Harradine | Self-ligating brackets and treatment efficiency[END_REF][START_REF] Turnbull | Treatment efficiency of conventional vs self-ligating brackets: effects of archwire size and material[END_REF][START_REF] Paduano | Time efficiency of self-ligating vs conventional brackets in orthodontics: effect of appliances and ligating systems[END_REF]. This difference was however supported in this meta-analysis by one single RCT [START_REF] Miles | Porcelain brackets during initial alignment: are self-ligating cosmetic brackets more efficient?[END_REF] including a limited number of patients. This clear clinical advantage was however substantiated by three former retrospective studies . The evidence behind the pain difference perceived by our patients when inserting or removing a rectangular wire, draws the same criticism. The advantage in favour of CB's in that matter is based on only one RCT [START_REF] Fleming | Pain experience during initial alignment with a self-ligating and a conventional fixed orthodontic appliance system[END_REF] of small sample size. This network meta-analysis allowed us to compare distinctively the thee bracket types two by two, unlike a former publication where all self-ligating brackets were pooled together and compared to CB's [START_REF] Wagner | Are self-ligating brackets more efficient than conventional brackets ? A meta-analysis of randomized controlled and split-mouth trials[END_REF]. Only two differences were identified between PSLBs and ASLBs:

• alignment was 10 days faster with ASLBs compared with PSLB's even if overall treatment duration between ASLBs and PSLBs was not significantly different • IMPA was greater with PSLBs compared with ASLB's in extraction cases (0.9° mean difference).

This faster initial alignment obtained with ASLB's compared to PSLB's corroborates the findings of Yang in 2017 [START_REF] Yang | Differences between active and passive self-ligating brackets for orthodontic treatment: systematic review and meta-analysis based on randomized clinical trials[END_REF].

Conclusion

The vast majority of the studied variables did not show any significant differences between the 3 types of brackets: active self-ligating brackets, passive self-ligating brackets and conventional brackets.

Results that were of statistical and clinical significance were the following: It was 5 (with assistance) to 6 (without assistance) times quicker to untie and ligate an 0.014 NiTi arch from/in 6 ASLBs anterior ceramic brackets compared to 6 ceramic CBs.

It was more painful to insert and remove an 0.019×0.025 SS wire in/from PSLB's brackets compared to CB's attachments.

The major difference between ASLBs and PSLBs was that alignment was 10 days faster with active self-ligating braces compared with passive self-ligating braces even if treatment duration between ASLBs and PSLBs was not significantly different. Most of the claims put forward by the suppliers were not substantiated by this network metaanalysis.

Conflicts of interest:

Authors declare that they have no conflict of interest regarding the materials cited in this paper.

Contribution

Order of authors: Raphaëlle Maizeray, Yves Bolender, Delphine Wagner, Hélène Levy-Bénichou, François Lefebvre (statistician).

LEGENDS OF FIGURES AND TABLES

Figure 1 :

 1 Direct comparison between outcome and treatment duration (months) of: a: PSLBs and CBs; b: ASLBs and CBs. (Active Self-Ligating Brackets, Passive Self-Ligating Brackets and Conventional Brackets.) Figure 2: Indirect comparison between PSLBs and ASLBs performed by network metaanalysis, outcome : treatment duration (months) Figure 3: PRISMA flow-chart Figure 4: Direct comparison between ASLBs and CBs of outcome: time to untie and ligate an arch wire. a: with assistance (seconds); b: without assistance (seconds).

Figure 5 :

 5 Direct comparison between PSLBs and CBs of outcome: experience of pain (on a visual analogue scale of 100). a: during insertion of an arch in SS 0.019x0.025; b: during removal of an arch in SS 0.019x0.025.

Figure 6 :

 6 Direct comparison between PSLBs and CBs of outcome: modification of maxillary incisor inclination in non-extraction cases (based on NA).

Figure 7 :

 7 Direct comparison between PSLBs and CBs of outcome: bleeding on probing (after 4-5 weeks) Figure 8: Direct comparison of outcome : mandibular alignment efficiency (days). a: between PSLBs and CBs; b: between ASLBs and CBs; c: between ASLBs and PSLBs.

Figure 9 :

 9 Direct comparison of outcome : IMPA change in lower first premolar extraction cases (degrees). a: between PSLBs and CBs; b: between ASLBs and CBs; between PSLBs and ASLBs.

Figure 10 :

 10 Direct comparison between PSLBs and CBs of outcomes : a: change in mandibular inter-molar width (mm); b: mandibular inter-canine width (mm).

Table I :

 I Characteristics of included studies Table II : Risk of bias assessment

	Studies Scott et al. Pringle et Oz et al. 2008 al. 2009 2012 [56] [25] [30] Miles 2006 [52] Pandis et al. 2006 [53] Pandis et al. 2006 [22] Pandis et al. 2007 [23] Scott et al. 2008 [24] Fleming et al. 2009 [27] Fleming et al. 2009 [26] Fleming et al. 2009 [29] Pandis et al. 2010 [31] Fleming et al. 2010 [28] Burrow 2010 [54] Miles and Weyant 2010 [32] Cattaneo et al. 2011 [33] Mezomo et al. 2011 [55] DiBiase et al. 2011 [34] Songra et Wong et al. 2013 [38] al. 2014 [42] da Costa Monini et Fleming et al. 2013 [39] al. 2014 [58] Othman et al. 2014 Dominguez and Velásquez 2013 [57] [43] Pandis et al. 2011 [35] Leite et al. 2012 [36] Atik et al. 2016 [44] Johansson and Lundström 2012 [37] Folco et al. 2014 [45] Cattaneo et al. 2013 [40] Reddy et al. 2014 [41] Nalçacı et al.	Study design RCT RCT Prospective and non randomized SMD Non randomized SMD Non randomized SMD RCT RCT RCT RCT RCT RCT RCT RCT Randomized SMD RCT RCT Randomized SMD RCT RCT RCT Randomized SMD RCT RCT Randomized SMD RCT RCT RCT RCT RCT RCT RCT	Participants characteristics: number; sex M/F; average age 60: 30/30 52: 24/28 19 (5 patients 16,27 ± 4,47 SL 15,2 ± 6,8 have only one CL 16,1 ± 7,4 maxillary premolar 58: 18/40 16,3 62 : 23/39 14 (13-16 years old) 105: 36/69 SLBs: 16,64 ± 3,54 CBs: 15,63 ± 2,09 54: 11/43 SLBs : 13,48 ± 1,31 CBs 13,92 ± 1,43 60: 31/29 16,27 ± 4,47 48: 16/32 15,96 ± 2,56 65: 22/43 16,28 ± 2,68 60: 21/39 16,35 ± 2,73 66 :27,32/38,68 Damon13,8(1,8) InOvation 13,8±1,7 54: 18/36 15,81 ±2,58 43: 24/19 14,8 ±6,24 60: 22/38 13,5 ±1,5 41 : ? PSL : 16 ±5,7 ASL : 15 ±3,3 15: 5/10 18 48 SL 16,19 ±3,38 CL 16,38 ±5,28 extraction): 5/14 98 : 36/62 13,6 40: 17/23 SLBs: 13,9 CBs and elastomeric ligatures: 14.1 ASLBs In-OvationR®: F: 13,8; H :14,1 PSLBs Damon 3MX®: F: 14,2; M: 14,3 CBs and Super Slick® CBs: F: 13,2; Low M :14,3 Friction 25 : 9/16 elastomeric 23,32 ±5,08 ligatures: 13,7 96 (49/47) Damon Q®: 18,9 ±2,9 In-Ovation C®: 22.5 ±8,5 CL 18.6 ±3.4 60 : 29/31 CBs : 18,3 59 : 19/40 ASL : 24,3 ±3,2 PSL : 24,3 ±3,1 ASLBs:18,9 PSLBs:17,6 50: 17/33 13.3 ±1.6 19 : 8/11 20,6 (11-30) 33: 0/33 SLBs: 14,8 CBs: 14,5 90: 26/64 SLBs : 15,3 (1,7) CBs : 15,0 (1,37) 22 : 11/11 46 : ? PSLBs :15,5 ±6 ASLBs : 14,4 ±3,3 48: 26/22 16,69 ±0,63	Intervention: type of brackets, brands of brackets (number of participants) PSLBs PSLBs PSLBs PSLBs Damon 3® Damon 3® SmartClip® SmartClip® (33) (24) (19) (9) PSLBs Damon 2® (58) PSLBs Damon 2® (43) PSLBs Damon 2® (52 patients among which 27 treated without extraction) PSLBs Damon 2® (27) PSLBs Damon 3® (32) PSLBs SmartClip® (26) PSLBs SmartClip® (32) PSLBs SmartClip® (29) ASLBs InOvationR® (33) PSLBs SmartClip® (28) SLBs (43): PSLBs Damon 3® (21) ASLBs SmartClip® (22) ASLBs In-Ovation C® (30) PSLBs Damon® (21) PSLBs SmartClip® (15) PSLBs Damon 3® (27) SLBs (78): PSLBs Damon 3MX® (14) ASLBs In-Ovation R® (37) PSLBs Damon 3MX® (41) ASLBs InOvationR® SLBs (59): PSLBs Damon Q® (28) ASLBs In-(25) ASLBs Ovation InOvationR® C® (31) ASLBs InOvationC® (29) (20) PSLBs Damon 3MX® (20) PSLBs Damon MX® (25) PSLBs Easyclip® (11) PSLBs Damon 3MX® (16) PSLBs Time 2® (44) PSLBs Damon 3® ASLBs InOvationR® (20) (11) SLBs (19) ASLBs In-Ovation R® (10)	Comparison: type of CBs CBs CBs Mini Uni-ligatures and 9 Synthesis® TruStraight® Twin® with with stainless (27) with stainless steel steel ligatures) brackets, brands elastomeric ligature on the of brackets (number of participants) CBs Victory® and elastomeric ligatures (58) CBs Microarch® (19) CBs Microarch® (53 patients among which 27 treated without extraction) CBs Microarch® (27) with elastomeric ligatures CBs Synthesis® (28) with elastomeric ligatures CBs Victory® (22) CBs Victory® (33) with elastomeric ligatures ligatures (28) CBs Victory® (31) PSLBs Damon 3MX® (33) CBs Victory® (26) CBs Victory® canine and stainsless steel ligature (43) CBs Clarity® and elastomeric ligatures (30) elastomeric ASLBs InOvation® (20) CBs Gemini® elastomeric ligatures (15) CBs Synthesis® avec ligatures élastomériques (21) canine (19) CBs Omni® CBs (26) with elastomeric ligatures with elastomeric ligatures (20) CBsOvation® (25) CBs Ovation® with elastomeric ligatures (28) CBs Victory series® (20) CBs Equilibrium® (30) CBs Microarch® (25) CBs 3M unitek, Monrovia, Calif® (8) CBs Forestadent® + QHX (17) CBs Gemini® (46) CBs Synthesis® PSLBs Damon 3MX® (26) with elastomeric ligatures (11) CBs (29 among which 10 patients with	Results Pain Pain (Irregularity Maxillary Mandibular perception (4 perception (4 Index) space closure incisor hours, 24 hours, 24 rate inclination hours, 3 days, hours, 24 (mm/month) change Initial alignment rate between mandibular canines (Irregularity Index) Pain perception at chairside during appliance manipulation and after. Bond failure Bond failure Maxillary incisor inclination modification Mandibular alignment rate from canine to canine Mandibular incisor inclination modification Mandibular transverse dimension changes (models) 7 days after appliance placement) Use of painkillers Initial alignment rate between mandibular canines Mandibular arch dimension modifications (models) Mandibular alignment rate from canine to canine Mandibular central incisor root resorption Pain perception (4 hours, 24 hours, 3 days, 7 days after appliance installation) Use of painkillers Pain perception at chairside Initial alignment rate between mandibular canines (Irregularity Index) hours, 3 days, 7 days after appliance installation) Mandibular arch dimensions modifications (models) Mandibular incisor inclination change Maxillary alignment rate from canine to canine Treatment duration Number of appointments Initial and final scores of Peer Assessment Rating (PAR) Canine retraction velocity Discomfort Time to tie and untie 6 maxillary anterior ceramic braces Mandibular initial closure alignment rate Maxillary transverse dimension modifications: between canines, between premolars, between molars (CBCT) Time for mandibular space closure (mm/month) Treatment duration Number of appointments Occlusal results (Peer Assessment Alignment Velocity average efficiency Rate of space of closure space closure on a 3-month period With 4 first premolars Maxillary extractions: Rate of space transverse closure dimension Loss of modification anchorage (models) OHRQoL: Oral Health Pain Related Quality of perception (4 hours, Life 24 hours, 3 days, Rating) Transverse dimension modifications (models) Incisor root resorption after 6 month of treatment 7 days after Maxillary and appliance mandibular placement) incisor Visual Analog position Scale (VAS) change after 2 hours, Maxillary 6 hours, 24 hours transverse and dimension 2,3,7 days of change application (models) (maxillary (CBCT) Treatment duration Number of appointments Treatment results (Index and Visible mandibular Plaque Index scores) Mandibular (VP) Gingival incisor Bleeding inclination Index (GBI) change Probing of Mandibular Depth (PD) Complexity, Outcomes and Needs ICON) alignment efficiency and passive space	Low Low High Risk of bias High High Moderate Moderate Low Low Low Low Low Low Moderate Moderate Low Moderate Low Low Low Low Low Low Low Moderate Low High Low Moderate Moderate Low

[START_REF] Nalçacı | Effect of bracket type on halitosis, periodontal status, and microbial colonization[END_REF] RCT


Bartlomiej Szabat-Iriaka

Marc Le Borgne
email: marc.le-borgne@uni-lyon1.fr

Brain safety concerns of nanomedicines: the need for a specific regulatory framework

Keywords: nanomedicines, new therapeutic entities, mRNA vaccines, nanocarriers, BBB crossing, protein corona, toxicity, regulatory framework, FDA, EMA

Teaser: mRNA-lipid nanoparticle vaccines have been authorized in recent months. These nanomedicines can undergo in vivo a phenomenon called protein corona, which could affect their biodistribution. Current regulations evaluating their brain toxicity are still limited.

There is growing interest in using nanomaterials as carriers for the delivery of drugs in diseases such as cancers and central nervous system (CNS) disorders. Although several nanomaterial-based products have been approved, the regulatory framework for their use in humans remains limited. Nanomedicines (NMs) are usually not designed to cross the blood-brain barrier (BBB). Given the lack of a comprehensive set of standardized methods to assess their in vivo fate, there is an urgent need to characterize NM biodistribution as well as the toxicity that could result from their interaction with the CNS. Here, we discuss the risks of potential unwanted BBB crossing and brain toxicity of nanocarriers (NCs), along with the safety assessment and current regulatory challenges related to NMs.

Introduction

The use of NCs in human medicine is part of NM vectorization strategies designed to facilitate the passage of diagnostic and therapeutic agents through biological structures such as the BBB. [START_REF] Singh | Targeted therapy in chronic diseases using nanomaterial-based drug delivery vehicles[END_REF] NMs are usually formed via the encapsulation, trapping, or adsorption of molecules of interest within a lipidic, polymeric, or inorganic NC less than 100 nm in size. [START_REF] El-Say | Polymeric nanoparticles: promising platform for drug delivery[END_REF] NC surface functionalization, allowing tissue-specific drug delivery, can be used to target brain tumors, such as glioblastomas. [START_REF] Gregory | Systemic brain tumor delivery of synthetic protein nanoparticles for glioblastoma therapy[END_REF] or to treat neurological disorders, such as Alzheimer's disease or Parkinson's disease. [START_REF] Tomitaka | Surface-engineered multimodal magnetic nanoparticles to manage CNS diseases[END_REF] Nevertheless, targeted drug delivery does not exclude that NMs might be directed toward regions of the body in which their localization is unwanted. [START_REF] Alexander | Recent expansions of novel strategies towards the drug targeting into the brain[END_REF][START_REF] Sim | Nanoparticle-based technology approaches to the management of neurological disorders[END_REF] This might be concerning for therapies or diagnostic agents not designed to be located in the brain, especially when they are intended to be used in vulnerable populations, such as children or older patients. Although NMs can be delivered through different routes, such as intranasal administration. [START_REF] Islam | Intranasal delivery of nanoformulations: a potential way of treatment for neurological disorders[END_REF] they remain administered mainly intravenously. Therefore, NMs need to go through the BBB to access the brain. This barrier, which covers 99% of the surface of cerebral capillaries, acts as a selective filter blocking between 98% and 100% of micro-and macromolecules, respectively. [START_REF] Zhang | Strategies for transporting nanoparticles across the blood-brain barrier[END_REF]9 NM biodistribution is influenced by various factors that also have a role in the passage of NMs through the BBB. [START_REF] Kaga | Influence of size and shape on the biodistribution of nanoparticles prepared by polymerization-induced self-assembly[END_REF][START_REF] Jo | Size, surface charge, and shape determine therapeutic effects of nanoparticles on brain and retinal diseases[END_REF][START_REF] Sempf | Adsorption of plasma proteins on uncoated PLGA nanoparticles[END_REF] Even though several NMs have already been approved, the in vivo fate and BBB interaction of these systems need to be further investigated, given the increasing and diversifying number of applications using NCs (Table 1). [START_REF] Anselmo | Nanoparticles in the clinic: an update[END_REF][START_REF] Damasco | Understanding nanoparticle toxicity to direct a safe-by-design approach in cancer nanomedicine[END_REF][START_REF] Editorial | Nanomedicine and the COVID-19 vaccines[END_REF] Over the past few years, initiatives have been undertaken to provide specific evaluation criteria for this new class of drug. These initiatives have established initial methodological and regulatory frameworks, but these remain incomplete and mostly focused on lipid NCs. [START_REF]Liposome drug products: Chemistry, manufacturing, and controls; human pharmacokinetics and bioavailability; and labeling documentation[END_REF][START_REF] Fda | Drug products, including biological products, that contain nanomaterials[END_REF] The importance of the distribution of NCs within the organism and the ability of these systems to interact with biological barriers, such as the BBB, require better characterization of both the mechanisms used to pass into the brain and the influence of biological environments on NC behavior.

Nanomedicines, nanocarriers, and the blood-brain barrier

The brain has a central role in the cognitive development of individuals, even though it accounts for only 2% of the total body mass. [START_REF] Alexander | Recent expansions of novel strategies towards the drug targeting into the brain[END_REF] The passage of molecules from the bloodstream to the brain depends on three barriers: BBB, blood cerebrospinal fluid barrier (BCSFB); and blood-arachnoid barrier (BAB). [START_REF] Vendel | The need for mathematical modelling of spatial drug distribution within the brain[END_REF] Given that the BBB is the main barrier among the three, it explains why cerebral capillaries, which account for 85% of the vascular network of the brain, are mainly lined with BBB endothelial cells. [START_REF] Zhang | Strategies for transporting nanoparticles across the blood-brain barrier[END_REF][START_REF] Vendel | The need for mathematical modelling of spatial drug distribution within the brain[END_REF][START_REF] Sweeney | Blood-brain barrier: from physiology to disease and back[END_REF] These cells, which form an inner layer connected by tight junctions, are completed by an outer layer that includes a basement membrane, astrocytes, pericytes, and neurons. [START_REF] Alexander | Recent expansions of novel strategies towards the drug targeting into the brain[END_REF][START_REF] Zhang | Strategies for transporting nanoparticles across the blood-brain barrier[END_REF][START_REF] Sweeney | Blood-brain barrier: from physiology to disease and back[END_REF] The BBB forms a selective barrier that limits the passage of molecules to the cerebral parenchyma. NMs can cross the BBB in different ways. In targeted vectorization, intracellular uptake depends mainly on receptor-mediated transcytosis. [START_REF] Ding | Overcoming blood-brain barrier transport: sdvances in nanoparticle-based drug delivery strategies[END_REF] For some inorganic NCs, such as gold nanoparticles (AuNPs), passive transcellular diffusion might also occur. [START_REF] Gromnicova | Glucose-coated gold nanoparticles transfer across human brain endothelium and enter astrocytes in vitro[END_REF] In addition, inorganic NCs can facilitate paracellular diffusion transport by disrupting the tight junctions of endothelial cells. [START_REF] Li | Gold nanoparticles increase endothelial paracellular permeability by altering components of endothelial tight junctions, and increase blood-brain barrier permeability in mice[END_REF][START_REF] Liu | Blood-brain barrier dysfunction induced by silica NPs in vitro and in vivo: Involvement of oxidative stress and Rhokinase/JNK signaling pathways[END_REF] Paracellular diffusion is usually only present on 0.006% of the BBB surface. [START_REF] Yamamoto | Predicting drug concentration-time profiles in multiple CNS compartments using a comprehensive physiologically-based pharmacokinetic model[END_REF] The disorganized vascular architecture of tumors implies the existence of large fenestrations between endothelial cells. [START_REF] Greish | Enhanced permeability and retention effect for selective targeting of anticancer nanomedicine: are we there yet?[END_REF] This leaky vasculature allows NMs to cross the BBB through the openings between endothelial cells via a phenomenon known as the enhanced permeation and retention effect. [START_REF] Greish | Enhanced permeability and retention effect for selective targeting of anticancer nanomedicine: are we there yet?[END_REF][START_REF] Du | Facile marriage of Gd 3+ to polymer-coated carbon nanodots with enhanced biocompatibility for targeted MR/fluorescence imaging of glioma[END_REF][START_REF] Umlauf | Exploiting BBB disruption for the delivery of nanocarriers to the diseased CNS[END_REF] The ability of NMs to cross the BBB is influenced by the size, shape, and charge of the NC, as well as the functional groups on its surface. [START_REF] Agrahari | Nanoformulation properties, characterization, and behavior in complex biological matrices: challenges and opportunities for brain-targeted drug delivery applications and enhanced translational potential[END_REF] Certain biological phenomena, such as the protein corona (PC), can also influence BBB crossing by NMs. [START_REF] Qi | In vivo fate of lipid-based nanoparticles[END_REF] The adsorption of serum proteins at the NC surface, also known as the PC, can facilitate the passage of NMs through the BBB. [START_REF] Dal Magro | Artificial apolipoprotein corona enables nanoparticle brain targeting[END_REF] PC formation is a dynamic phenomenon that involves a change in the type of protein adsorbed at the NC surface, based on the circulation time and interaction with the immediate biological environment (Figure 1). 31,[START_REF] Nel | Understanding biophysicochemical interactions at the nano-bio interface[END_REF] The composition of the PC also varies with the size and surface charge of the NC. [START_REF] Nel | Understanding biophysicochemical interactions at the nano-bio interface[END_REF] Table 2 provides a list of proteins that can be found in the PC. Indeed, some of these proteins might be common to several NC categories, whereas others can only be found in one type of NC. 33 Some of these proteins, such as human transferrin or apolipoproteins, are known to cross the BBB. [34][35][START_REF] Zhou | Apolipoprotein A-I crosses the blood-brain barrier through clathrin-independent and cholesterol-mediated endocytosis[END_REF] Additionally, there may be preferential adsorption of proteins specialized in transport through the BBB, such as afamin, on certain categories of functional groups used for targeting. 33 In active transport, such as transferrin receptor-mediated endocytosis, different intracellular trafficking routes can be followed by NMs. They can either cross the entire BBB or be redirected toward different intra-endothelial compartments for further processing. [START_REF] Clark | Increased brain uptake of targeted nanoparticles by adding an acid-cleavable linkage between transferrin and the nanoparticle core[END_REF][START_REF] Ko | Nanoparticle-mediated delivery of oligonucleotides to the blood-brain barrier: In vitro and in situ brain perfusion studies on the uptake mechanisms[END_REF] The potential diffusion of NMs in brain parenchyma should raise questions about the risks associated with the localization of NMs not intended for the CNS, given the large number of applications using NCs and which might involve vulnerable populations (Table 1).

Thus, it becomes obvious that, during the development of a NM [quality (Q) in manufacturing, preclinical safety (S) tests, and clinical efficacy (E) studies], the manufacturer must be able to combine different analytical methods to also study its NM in complex biological environments. It is similar to collecting Q/S/E data for the future Common Technical Document (CTD) in a real situation, without excessive approximations. Min et al. recently reviewed the tools used to explore the PC phenomenon observed with NMs, such as transmission electron microscopy (TEM), surface-enhanced Raman scattering, and small-angle X-rays scattering. [START_REF] Huang | Research progress and application opportunities of nanoparticle-protein corona complexes[END_REF]

Brain safety concerns

The range of potentially detrimental effects on the brain caused by NMs reflects the diversity of structures used for their vectorization. Cationic lipid NPs (LNPs) are frequently used as drug delivery systems because they can enter cells more easily compared with anionic NCs. [START_REF] Fda | Nanotechnology: over a decade of progress and innovation at FDA[END_REF][START_REF] Ho | Nano-cell interactions of non-cationic bionanomaterials[END_REF] However, cationic LNPs are in general more toxic than anionic LNPs. [START_REF] Ho | Nano-cell interactions of non-cationic bionanomaterials[END_REF] Positively charged LNP are likely to increase brain vascular volume, to cause BBB damage, and potentially lead to the formation of cerebral edema. [START_REF] Lockman | Nanoparticle surface charges alter blood-brain barrier integrity and permeability[END_REF]43 Some NMs, such as mRNA vaccines, contain optimized LNPs with an ionizable surface charge that is supposed to remain neutral at physiological pH. [START_REF] Hassett | Optimization of lipid nanoparticles for intramuscular administration of mRNA vaccines[END_REF] However, it cannot be ruled out that this surface charge might change in individuals with conditions that can cause pH variations. The surface charge of nanocarriers has an influence on NM biodistribution, but biological phenomena, such as PC, can also influence their in vivo fate. [START_REF] Qi | In vivo fate of lipid-based nanoparticles[END_REF][START_REF] Grabbe | Translating nanoparticulate-personalized cancer vaccines into clinical applications: case study with RNA-lipoplexes for the treatment of melanoma[END_REF] Indeed, the PC can facilitate the passage of NMs through the BBB. [START_REF] Rehman | Blood-brain barrier amenable gold nanoparticles biofabrication in aged cell culture medium[END_REF] The nature of the PC can differ before and after NM passage through the BBB. 47 The evolution of the PC composition during transport through endothelial cells suggests that proteins that are not initially found in the cerebral parenchyma might enter and disrupt brain homeostasis. 47 In addition, some metabolic imbalances, such as altered autophagy or lysosomal activation, can be observed in endothelial cells or in the CNS after inorganic NC interactions with the BBB. [START_REF] Chang | Neurotoxicity of metal-containing nanoparticles and implications in glial cells[END_REF] Autophagy is a mechanism that might be involved in the development of neurodegenerative diseases, such as Alzheimer's disease or Parkinson's disease. [START_REF] Plaza-Zabala | Autophagy and microglia: novel partners in neurodegeneration and aging[END_REF] Oxidative stress resulting from NPs has been identified as one possible mechanism for the disruption of autophagy. [START_REF] Jia | Nanoparticles induce autophagy via mTOR pathway inhibition and reactive oxygen species generation[END_REF] Inorganic NCs used in therapeutics are often responsible for the production of reactive oxygen species involved in oxidative stress mechanisms. [START_REF] Jia | Nanoparticles induce autophagy via mTOR pathway inhibition and reactive oxygen species generation[END_REF] For example, silver NPs can generate a significant oxidative stress, which can cause neurotoxicity. [START_REF] Khan | Silver nanoparticle-induced expression of proteins related to oxidative stress and neurodegeneration in an in vitro human blood-brain barrier model[END_REF] Thus, toxicity risks resulting from NM interactions with the BBB are not negligible. Given the lack of specific methods for investigating the central toxicity of NMs, especially in vivo, it is important that the regulation provide a standardized methodological framework, which will help to better characterize the behavior of these NMs in different biological environments.

Regulatory challenges

Since 1970, more than 600 applications for nanomaterials have been submitted to the US Food and Drug Administration (FDA), with an increase in submissions over the past 10 years. [START_REF] Fda | Nanotechnology: over a decade of progress and innovation at FDA[END_REF] Although several NMs are already on the market, there is still no specific regulatory framework for the study of their toxicity in human. [START_REF] Sainz | Regulatory aspects on nanomedicines[END_REF][START_REF] Kad | An elucidative review to analytically sieve the viability of nanomedicine market[END_REF] The main challenge faced by regulatory agencies is the development of an appropriate classification that includes the variety of platforms used for the development of nanomaterials for preventive, therapeutic, and diagnostic purposes. [START_REF] Paradise | Regulating nanomedicine at the food and drug administration[END_REF] During the second half of the 2000s, international bodies, such as the FDA, the European Commission, and the Organisation for Economic Co-operation and Development (OECD), undertook initiatives to standardize the safety evaluation process of drug products containing nanomaterials. In particular, they issued several works that support the development of NMs. [START_REF]Nanotechnology guidance documents[END_REF][56][57] For example, under the NANoREG project, the Joint Research Centre from the European Commission proposed to manufacturers and regulators an overview of available tools (e.g., test methods and decision trees) for the safety assessment. [START_REF]NANoREG toolbox for the safety assessment of nanomaterials[END_REF] Among all documents made available by the FDA. [START_REF]Nanotechnology guidance documents[END_REF] there is the guidance referring to 'drug products, including biological products, that contain nanomaterials'. [START_REF] Fda | Drug products, including biological products, that contain nanomaterials[END_REF] As it notes, the biological fate of the NC and its potential impact on safety might need to be assessed in addition to those of the active pharmaceutical ingredient because of a risk of increased crossing of biological barriers. Therefore, it becomes necessary to realize in vivo biodistribution studies using labeled nanomaterials in animals. Regarding clinical development, further nonbinding recommendations are listed, such as the evaluation of pharmacokinetic studies of the NC. [START_REF] Fda | Drug products, including biological products, that contain nanomaterials[END_REF] Although these recommendations are not specific to the study of brain toxicity for brain-and nonbrain-targeting NMs, several insights can be derived from existing documents to identify potential impacts on brain structures. For example, a mass balance study of a drug substance labeled with a radioactive isotope in a liposomal formulation and in a nonliposomal formulation could be completed by confocal microscopy and TEM to assess qualitatively and quantitatively the distribution of NM at subcellular level. [START_REF]Liposome drug products: Chemistry, manufacturing, and controls; human pharmacokinetics and bioavailability; and labeling documentation[END_REF] Likewise, in the case of drugs containing nanomaterials developed from a reference drug product, the use of single and multiple dose pharmacokinetic-pharmacodynamic studies for medium and high-risk categories should provide further indication of tolerability compared with a simple bioequivalence study. [START_REF] Gottardo | NANoREG framework for the safety assessment of nanomaterials[END_REF] This transposition of generic procedures into a particular setting helps to increase specificity, but there are still limitations.

Therefore, the applicability of traditional analytical methods to all NMs remains questionable. For example, although guidelines recommend the use of at least two different analytical techniques to characterize in vitro and in vivo NMs, there are still no conventional or alternative analytical techniques validated specifically for NMs. Thus, NM marketing authorization mainly relies on traditional evaluation methods that are not well suited to take into account the specific characteristics of NMs. [START_REF] Farjadian | Nanopharmaceuticals and nanomedicines currently on the market: challenges and opportunities[END_REF] In addition, as previously reported, the PC confers singular properties to NMs. Current guidelines provide limited insights on how to assess NM behavior in vivo.

In July 2020, the FDA published a new report retracing the history of the progress as well as the commitments undertaken over the past two decades, but this document has answered questions on how to address the lack of specificity of NM toxicity assessment. [START_REF] Fda | Nanotechnology: over a decade of progress and innovation at FDA[END_REF] These elements underline the importance of quickly defining an appropriate regulatory framework able to respond to the safety challenges of NMs, especially with the arrival of NMs intended for preventive applications, such as mRNA vaccines. [START_REF] Editorial | Nanomedicine and the COVID-19 vaccines[END_REF] The efforts undertaken by the FDA, the European Union, and the OECD must lead to the standardization of the safety validation processes to reduce the use of a case-by-case approach. [START_REF] Sainz | Regulatory aspects on nanomedicines[END_REF]

Concluding remarks

The variety of approaches used in the development of NMs in human health complicates the implementation of a rational classification of these drugs and the elaboration of a normative framework that can respond to the issues related to their use in humans. This issue, combined with the lack of specific methods for evaluating the in vivo fate of NM, should encourage regulatory authorities to consider the challenges regarding the safety of products on the market or in development. The magnitude of the current Coronavirus 2019 (COVID-19) health crisis has prompted governments and industrials to quickly find solutions capable of curbing the spread of severe acute respiratory syndrome coronavirus 2 (SARS-Cov-2). However, the main consequence of the global rush has been a dramatic shortening of preclinical and clinical development times as well as an increase in the use of emergency approval procedures (e.g., rolling review by the EMA and emergency use authorization request to the FDA).

Part of the response to stop the pandemic includes the use of vaccine categories for which LNP serve as drug delivery systems. Apart from the massive use of NMs in an unprecedented health crisis, the need to define the evaluation criteria specific to this new category of products is explained by the arrival of NMs designed for the prevention and treatment of vulnerable populations, such as infants or older patients, for whom the undesired location of NMs in the brain could have a significant clinical impact. In vivo protein corona on nanoparticles. After administration, bare spheres can gather serum proteins and then forming nanoparticle-protein corona complexes in the blood stream. After crossing the blood-brain barrier (BBB), qualitative and quantitative changes of corona are observed. The evolution of the percentages of the four proteins after crossing the BBB was calculated from 47 .

In vivo protein corona on nanoparticles. After administration, bare spheres can gather serum proteins and then forming nanoparticle-protein corona complexes in the blood stream. After BBB crossing, qualitative and quantitative changes of corona are observed. After the passage of the BBB, the evolution of the percentages of the four proteins was calculated from the reference [47].

Figure 1 .

 1 Figure 1.In vivo protein corona on nanoparticles. After administration, bare spheres can gather serum proteins and then forming nanoparticle-protein corona complexes in the blood stream. After crossing the blood-brain barrier (BBB), qualitative and quantitative changes of corona are observed. The evolution of the percentages of the four proteins after crossing the BBB was calculated from47 .

a

  Abbreviations: AmB, amphotericin B; MA, market authorization; MM, multiple myeloma; VL, visceral leishmaniasis. b This medicine received an emergency use authorization. c This medicine received a conditional marketing authorization (via a centralized procedure). d This medicine was approved via a national procedure.

Table 1 . Example of authorized/approved NMs in Europe and the USA

 1

	NM	Company	Particle type/active moiety	Indication	MA (year of issue)	Refs
	mRNA-1273	Moderna	LNP/mRNA	COVID-19	FDA b (2020); EU c (2020)	[15]
	BNT162b2	Pfizer/BioNTech	LNP/mRNA	COVID-19	FDA b (2020); EU c (2020)	[15]
	Patisiran; ALN-TTR02;	Alnylam Pharmaceuticals LNP/RNAi	Transthyretin-mediated	FDA (2018); EU (2018)	[13]
	ONPATTRO ®			amyloidosis
	CPX-351; VYXEOS ®	Jazz Pharmaceuticals	Liposome/cytarabine:daunorubicin Acute myeloid leukemia	FDA (2017); EU (2018)	[13]
	Paclitaxel; ABRAXANE ® Celgene	Albumin/paclitaxel	SNCLC, breast cancer,	FDA (2005); EU (2008)	[13]
				pancreatic cancer
	AmB; AmBisome ®	Gilead Sciences	Liposome/AmB	Fungal infections,	FDA (1997); France (1998) d	[13]
				cryptococcal meningitis, VL
	Doxorubicin; CAELYX ® Janssen	Liposome/doxorubicin	Ovarian cancer, HIV-	FDA (1995); EMA (1996)	[13]
				associated Kaposi's
				sarcoma, MM
	NBTXR3	Nanobiotix	Phosphate coating/hafnium oxide Soft-tissue Sarcomas	CE mark (2019)	[14]
	(radioenhancer);
	HENSIFY ®

Table 2 . Examples of proteins found in the PC a Proteins found in corona Preferred NC fixation (when available) b Refs

 2

Acknowledgments

The authors would like to thank Maena Le Borgne and Christine Janssen for the realization of the figure of the article.

Declaration of interests

The authors have no conflicts of interest to declare.