Jenifer Vega Rodriguez
email: jenifer-andrea.vega-rodriguez@gipsa-lab.fr

Nathalie Vallée
email: nathalie.vallée@gipsa-lab.fr

Thiago Chacon
email: thiago_chacon@hotmail.com

Christophe Savariaux
email: christophe.savariaux@gipsa-lab.fr

Silvain Gerber
email: silvain.gerber@gipsa-lab.fr

An Intra-and Inter-Dialectal Study of Korebaju Vowels

Keywords: Language description, Tucanoan, acoustic formant space, acoustic duration, dialectal variation

Korebaju (ISO 639-3 coe) [ḱóˀrèbàjɨ ] is an endangered Tukanoan language spoken in the foothills of the Colombian Amazon. Two fieldworks carried out between 2021 and 2023 on a sample of 24 native speakers (12 females and 12 males) from two different varieties: Tama and Korebaju, located in two different villages, provide new data for improving intra-and inter-dialectal phonetic-phonological description of Korebaju. This acoustic study focuses on the vowel system of each of these two varieties and is the first part of an ongoing project on vowel nasalization and glottalization in Korebaju. The acoustic and statistical analyses indicate that there are no significant interdialectal differences between vowels. However, differences between generations of the same sex and of the same variety have been evidenced in our analyses. These results also suggest that Korebaju speakers' perception of stronger glottalization in the Tama variant refers to a morphological distinction. However, this hypothesis is still being analyzed considering the language's tonal system, nasalization, and morphology.

Introduction

Korebaju is a tonal language spoken in the foothills of the Colombian Amazon, belonging to the western branch of the Tukanoan family. This community today is the result of the historical union of 4 distinct populations: Korebaju, Tama, Macaguaje, and Carijona, who have adopted Korebaju as their own language after their original language became extinct. However, these communities try to sustain their original culture through cultural diasporas. Likewise, cross-dialectal variation seems to be present in Korebaju as each of these communities has settled in different territories in the same geographic area, distinguishing themselves until today through their clan distribution [START_REF]Korebaju community, Proposal of Korebaju's pedagogical model[END_REF]. As claimed by the speakers of these communities, inter-dialectal variations exist and contribute to the identity factors of belonging to a clan. With a global population of around 2,000 native speakers, Korebaju is a definitely endangered language according to the UNESCO Word Atlas of Languages [START_REF]UNESCO, UNESCO Atlas of the World's Languages in Danger[END_REF]. The Tama (Lat 1.5945, Long -75.41448) and Korebaju (Lat 1.01744, Long -75.2914) communities are located one hour away from each other by canoe with a 15-HP motor, however, they share cultural events, organizational meetings in which all Korebaju communities also participate. The present investigation is part of a comparative study between two dialectal variants, Tama and Korebaju, which, according to the speakers, present distinctions not only at the prosodic level but also in the production and distribution of glottalization. Two more recent studies suggested that Korebaju has an inventory of six oral vowels /i, e, a, o, u, ɨ/, six nasal vowels /ĩ, ẽ, ã, õ, ũ, ɨ ̃ /, and three glottal vowels /aˀ/, /eˀ/ and /oˀ/ [START_REF] Rodriguez | The Vowel System of Korebaju[END_REF], as well as 17 consonants /p, t, k, pʰ, tʰ, kʰ, β, ɸ, s, h, w, c, m, n, ɲ, ʰɲ, r/, and a mixed segmental and suprasegmental glottalization system [START_REF] Rodriguez | Glottal Sounds in Korebaju[END_REF].

Previous analysis without considering dialectal variation

Before our investigation, few previous studies have described the vowels of Korebaju, and none have taken into account the dialectal variations: [START_REF] Dupont | La Langue Koreguaje (Tukano Occidental)[END_REF] proposed an inventory of 6 phonemic basic vowels /i, e, a, o, u, ɯ/, a suprasegmental nasalization based on nasal harmony, and suprasegmental glottalization as a consequence of a long vowel elision; [START_REF] Herrera Casimilas | Manual de pronunciación española para hablantes koreguajes basado en el análisis contrastivo a nivel fonológico de los dos idiomas[END_REF] advanced a 12-vowel system /i, e, a, o, u, ɯ, ĩ, ẽ, ã, õ, ũ, ɯ ̃/; finally, [START_REF] Cook | El idioma Koreguaje (Tukano Occidental)[END_REF] suggested a system with 6 basic vowels /i, e, a, o, u, ɨ/, a suprasegmental nasalization according to [START_REF] Dupont | La Langue Koreguaje (Tukano Occidental)[END_REF], and a glottal stop that is part of the consonant inventory. This last description pointed out the presence of two dialectal variants (Tama and Korebaju) but did not provide differential data between these two variants. [START_REF] Rodriguez | The Vowel System of Korebaju[END_REF], [START_REF] Rodriguez | Glottal Sounds in Korebaju[END_REF], [START_REF] Vega Rodriguez | Glottalisation en korebaju : la question d'un trait mixte segmental et suprasegmetal[END_REF] described the Korebaju (COE) variant with an inventory of six oral vowels including a high unrounded central vowel /i, e, a, o, u, ɨ/, six nasal vowels /ĩ, ẽ, ã, õ, ũ, ɨ / and three glottal vowels /aˀ, eˀ, oˀ/ as part of a mixed system of glottalization, segmental and suprasegmental, that would depend on the syllabic structure of the language as well as on the tonal contour of the preceding vowel in a CVV syllable.

Our recent investigation on the Korebaju variety

Previous studies on the Tama variety

Only one investigation to date has described the Tama (TAM) dialectal variant. [START_REF] Mora Cortés | Reconocimiento del pueblo Tama. Descripcion fonologica de su variante linguistica[END_REF] proposed a 11-vowel phonemic inventory, and nine corresponding allophones: [START_REF] Mora Cortés | Reconocimiento del pueblo Tama. Descripcion fonologica de su variante linguistica[END_REF] indicated that: (1) allophones of unrounded back closed vowels were present when a palatal consonant preceded them, as previously indicated by [START_REF] Rodriguez | The Vowel System of Korebaju[END_REF] for back and central closed vowels in the Korebaju variant; (2) the allophones of the mid vowels /e, o/ occurred when they preceded the rhotic [r] or succeeded it, or in stressed syllables; (3) finally, the nasal allophones were found in contexts of nasal consonants. However, no acoustic analyses that could support such phonemic assimilations were presented in this study. We propose, in the present investigation, an acoustic description of the vowel system of Korebaju with both cross-dialectal and social comparisons, by 2nd Annual Meeting of the ELRA/ISCA SIG on Under-resourced Languages (SIGUL 2023) 18-20 August 2023, Dublin, Ireland taking gender and two different generations as independent variables.

/i/ [j], /ĩ/, /e/ [ɛ], /ẽ/ [ɛ ], /a/, /ʉ/ [ɨ] [ɤ], /ʉ / [ɨ ], /u/ [w], /ũ/, /o/ [ɔ], /õ/ [ɔ ]. Moreover,

Method

Two fieldworks were conducted for this study. The first one carried out from December 2021 to March 2022, and the second one from December 2022 to February 2023. Our acoustic study was carried out by analyzing production data of native speakers recorded at normal speech rate. These data were obtained from a survey of the vowel system of the TAM and COE varieties, which included an elicitation paradigm based on minimal and quasi-minimal pairs.

Participants

24 native speakers (12 females and 12 males) divided equally in each dialectal variant TAM and COE (six males and six females), from two different generations (G1 from 18 to 31 years old, and G2 from 42 to 70 years old) were involved in the study. All participants were native speakers of Korebaju and were of Korebaju and Tama descent. They also spoke Spanish as a second language, with the local settlers and at the boarding secondary school of the region. No speaker had left the community for more than two weeks at the time the recording took place.

Materials

A D800 EGG electroglottograph from Laryngograph was used to gather synchronized sound, electroglottographic, nasal, and oral airflow The EGG was connected directly to a laptop computer through a USB port. VoiceSuite 10.4.0 software [START_REF][END_REF] was used for data collection. Praat software [START_REF] Boersma | Praat, a system for doing phonetics by computer[END_REF] was used for segmentation, transcription, and analyses of the collected data, and R for quantitative and statistical analyses [START_REF] Core | R: A language and environment for statistical computing[END_REF]. Recordings were made with an omnidirectional microphone placed inside the Oronasal chamber Teen-Adult mask from Glottal Enterprise and connected to the D800 EGG. The sampling frequency was 24 Khz for each of the four-channel recorded (i.e. wav, EGG, nasal and oral airflows). Recordings were made in an enclosed area and at certain times of the day to avoid background noise and atmospheric sounds of the Amazon rainforest, such as bird songs and other sounds produced by forest animals.

Procedure and data analysis

Two list of 118 and 145 words, embedded in a carrier sentence, were recorded between 2021 and 2023, respectively. The first list of 118 words was collected for all 24 speakers. This list was designed to obtain the production of minimal and quasiminimal pairs in as many word contexts as possible. The second list of 145 words was recorded for 12 speakers (three speakers of each variety from both genders and both generations). This second list was conducted in order to complete the identification of minimal and quasi-minimal pairs in all possible contexts among the speakers of each variety and to verify the existence of nasal harmony and tonal contours.

The carrier sentence was constructed as follow: /cɨ kɨ ńà ìkámè ___ kóˀrèbàhɨ ́ cɨ ópí/ {cɨ kɨ ńà ìká-mè ___ kóˀrèbàhɨ ́ cɨ ópí} we say-PL ___ Korebaju language 'We say____in Korebaju'

Measurements were performed through a manual segmentation of words, syllables, and phones, using for each vowel delimitation the beginning and the end of the stable part of the second formant F2. This task was followed by a fine phonetic and phonological labeling. Then, an automatic extraction of the f0 and of the first four formants at 30%, 50%, and 70% of the vowel duration was performed. Statistical analyses were done with different models. For the analysis of oral vowel formants, statistical tests were performed by using three different Generalized Linear Mixed Models (GLMM). For each response variable (F1, F2, F3), we studied the impact of the fixed factors DIALECT (COE and TAM), GENDER (F and H), GENERATION (G1 and G2), VOWEL (a, e, i, o, u, ɨ), PERCENTAGE (30, 50, 70) and their interactions. The factor PARTICIPANT was introduced as a random effect. These models allowed at the same time to take into account the repetition of the measurements, the residual variances that can change between the modalities of the same factor, and also the correlations of the values of the response variables between the percentages. We performed them using the lme function of the nlme package of the statistical software R.

In order to determine if glottalization is part of vowel quality or is a contiguous additional segment, we measured and compared the durations of glottal and non-glottal vowels. We took into account the variation of duration related to speech velocity by dividing vowel length by word length (variable Ratio, corresponding to proportional (or relative) vowel duration). We statistically tested the impact of the fixed factors DIALECT, GENDER, GENERATION, VOWEL, and their interactions, on the response variable i.e. vowel proportional duration (Ratio). We performed for this a beta regression with random effect [START_REF] Cribari-Neto | Zeileis Beta Regression in R[END_REF]. This model allowed us to take into account the repetition of the measurement (the factor PARTICIPANT was introduced as a random effect in the model), and the fact that the values of the response variable Ratio are by definition included in the interval [0,1]. For this, we used the glmmTMB function of the glmmTMB package of the R statistical software.

Once the models were established, we performed contrast analyses with the glht function of the multcomp package, according to the method presented by [START_REF] Bretz | Multiple comparison procedures in linear models[END_REF], and using the emmeans package to construct the contrast matrices.

The different figures of the acoustic signal, EGG, and spectrogram were extracted using the Praatfig script [START_REF] Nguyen | Script Praatfig[END_REF] and Visible Vowels software [START_REF] Heeringa | Visible Vowels: a Tool for the Visualisation of Vowel Variation[END_REF].

Results and discussion

Oral vowels

Our phonological survey corroborates the six oral vowels described by [START_REF] Rodriguez | The Vowel System of Korebaju[END_REF] /i, e, a, o, u, ɨ / for the two dialectal varieties.

Figure 1 presents the distribution of the set of oral vowels in the two F1-F2 formants acoustic space for female speakers from the two varieties Korebaju (COE) and Tama (TAM). A centralization of the mid-front unrounded vowel [e] is evident for both varieties resulting in a mid-central allophone [ə] which appeared to be in free variation. A centralization phenomenon is also observed among the productions of the high front unrounded /i/ for COE females.

A large acoustic variability is found for the production of the open vowel /a/ for all 24 speakers. A tendency of vowel backing in the production of /a/ is observed for all the participants. with a Lobanov normalization [START_REF] Lobanov | Classification of Russian vowels spoken by different speakers[END_REF].

Before palatal consonants, we noted a fronting of the high central unrounded vowel /ɨ/ which presents as a near-high front unrounded allophone [ɪ], and of the high back rounded vowel /u/ which is realized as a near-high back rounded vowel [ʊ] (Table 1). Note that his second allophone differs from the study previously conducted for the Korebaju variant [START_REF] Rodriguez | The Vowel System of Korebaju[END_REF].

Table 1: Mean (and SE) F1, F2, F3 values (Hz) for the allophones [ɪ] and [ʊ], for females and males of the two generations of the two varieties TAM and COE.

Globally, no significant difference between the two language variants was found; neither between the two generations of each variety. However, some significant differences were found between males and females of the same generation and the same variety. Moreover, a significant difference at all three points (30%, 50%, and 70%) of vowel duration between males and females is found for the first formant F1 for the vowels [a], [e], [i], [ɨ],

[o] of COE G2 as well as for vowel [a] for COE G1 and TAM G1; finally, a gender difference is evident for F1 for the vowel [u] of the TAM G2 (Table 2 and Figure 4).

Table 2: Z (and P) values for F1 measured at 50% of the vowel duration, between females and males of the two generations and varieties TAM and COE.

No significant difference was found between F2 and F3 formants for any vowel in any variety, gender or generation.

Figure 4: Mean F1 values (Hz) and confidence intervals measured at 30%, 50% and 70% of vowel duration for females and males TAM of the two generations.

Overall, the main F1 difference found between males and females of COE G2-speakers is +200 Hz for the set of the oral vowels /i, e, a, a, o, u, ɨ/. For TAM G1-speakers, the mean values of F1 present some differences from +120 Hz to +130 Hz, but only for the vowel /a/ since TAM G1 vowels have approximately more homogeneous formant values. Our investigation did not find either allophone for the midcentral vowels /e, o/ in rhotic context as described by [START_REF] Mora Cortés | Reconocimiento del pueblo Tama. Descripcion fonologica de su variante linguistica[END_REF] for the TAM variant. However, our study attests an allophone in free variation for the front unrounded vowel /e/ corresponding to the mid-central vowel [ə] (Table 2).

Glottal vowels

Analyses of vowel durations indicate no significant differences between non-glottal and glottal vowels of the COE variety supporting the results of previous study [START_REF] Rodriguez | The Vowel System of Korebaju[END_REF]. Figures 5 and6 show boxplots of the proportional duration (Ratio values) of modal and their corresponding glottal vowels of the TAM variety. Although there is a tendency for all glottal vowels to be longer in duration, this difference is not significant for the vowel set which suggests that either glottal vowels are phonemic in both varieties or glottalization is suprasegmental [START_REF] Stenzel | Glottalization and other suprasegmental features in Wanano[END_REF]. Note also the absence of glottal realizations of /u/ in TAM females, as said above. Our survey provided a set of 5 phonological glottal vowels for both language variants / iˀ, eˀ, aˀ, oˀ, ɨˀ/. A glottal back-rounded vowel [uˀ] was randomly produced by speakers of both generations and was surprisingly omitted by all females of the TAM variant. This phone can occur in any position but its phonological status has not yet been confirmed. Our investigation supports a phonological contrast between modal and glottal for the peripheral vowels, as proposed by [START_REF] Rodriguez | The Vowel System of Korebaju[END_REF], [START_REF] Rodriguez | Glottal Sounds in Korebaju[END_REF], and [START_REF] Vega Rodriguez | Glottalisation en korebaju : la question d'un trait mixte segmental et suprasegmetal[END_REF], through minimal pairs in first and second root positions as well as in some affixes. However, it also provides evidence for a phonological status for the high glottal unrounded vowels 'dirty Bactris Gasipaes'

Nasal vowels

Our examination of the acoustic structure of vowels, which included spectrographic analyses, showed that the nasal formant frequencies belong to the syllable containing the nasal vowel and not to the whole word. As a result, our investigation did not find a nasal harmony affecting the whole word. Nasality seems limited to the domain of the syllable.

c) [pi ã] [pi ʔa] d) [ma ̃̃̂:] [màʔ̪ á] 'chili' 'bird' 'macaw' 'road' f) [ci õ] [ci ʔo ] g) [cái ] [càʔí] 'girl' 'crop' 'jaguar' 'liane_yare'
Considering that Korebaju is a tonal language [3], [START_REF] Rodriguez | Glottal Sounds in Korebaju[END_REF], [START_REF] Dupont | La Langue Koreguaje (Tukano Occidental)[END_REF], [START_REF] Cook | El idioma Koreguaje (Tukano Occidental)[END_REF], [START_REF] Gralow | The coreguaje suprasegmental system: tone, stress and intonation[END_REF], and that the intervocalic glottal is still under investigation as there is no consensus on its segmental or suprasegmental status either in the previous descriptions of Korebaju nor in the descriptions in other languages of the Tukanoan family ( [START_REF] Sorensen | The Morphology of Tukano[END_REF], [START_REF] Klumpp | Sistema Fonológico del Piratapuyo[END_REF], [START_REF] Miller | Desano Grammar. Studies in the languages of Colombia, 6[END_REF], [START_REF] Vallejos | El secoya del Putumayo: aportes fonológicos para la reconstrucción del Proto-Tucano Occidental[END_REF], [START_REF] Bruil | Clause-typing and evidentiality in Ecuadorian Siona[END_REF], [START_REF] Stenzel | Glottalization and other suprasegmental features in Wanano[END_REF]), such word pairs cannot be categorized as minimal pairs that may distinguish phonemic oral and nasal vowels in Korebaju in either variety.

Conclusion

Our study did not find any interdialectal differences between TAM and COE. Intradialectal differences could be observed at the gender level for certain generations and certain vowels. This study confirms that glottalization seems to be part of the vowel as noted by [START_REF] Rodriguez | The Vowel System of Korebaju[END_REF] and [START_REF] Vega Rodriguez | Glottalisation en korebaju : la question d'un trait mixte segmental et suprasegmetal[END_REF], although its status as a segmental articulatory feature or a suprasegmental is yet unclear. We found a tendency for glottal vowels to be longer, but this difference was not significant for all set of vowels. Furthermore, this research found two phonemes corresponding to the closed glottal vowels /ɨˀ/ and /iˀ/.

Finally, this study suggests that the possible perceptual cause of a strong glottalization for speakers of the TAM variant may be due to a morphological change of certain words like the word 'narrow' where a possible insertion of a copulative predicate at the second syllable in the TAM variant creates the condition for a resyllabification of the word [má-ʔ̰ -àˀ-kàrɨ ] {CL-COP-narrow-CL}, while the COE variant will produce a modulated tone without insertion of the same copula [mâˀ-kàrɨ ] {CL-narrow-CL}.

These results are still under investigation. Our ongoing research is looking at the relationship between tone and nasalization and the different types of glottalization present in the language.

Figure 1 :

 1 Figure 1: F1/F2 distribution of the six oral vowels produced by 6 females of each variety (COE and TAM) with a Lobanov [17] normalization.

Figure 2

 2 Figure2displays the acoustic distribution in the F1/F2 space of the oral vowel set for the male speakers of the two varieties, COE and TAM. Centralized realizations of the front mid vowel /e/ appear as in the case of female speakers. The vowel fronting allophone of / ɨ / is less evident in males of the COE variant than in males of the TAM variant.

Figure 2 :

 2 Figure 2: F1/F2 distribution of the six oral vowels produced by 6 males of each variety (COE and TAM), with a Lobanov normalization [17].

Figure 3

 3 displays the differences in F1 for the vowels [a], [e], [i], [ɨ] [o], and [u], of the two generations of the COE variant taken at 30%, 50%, and 70% of vowel duration.

Figure 3 :

 3 Figure 3: Mean values (Hz) of F1 measured at 30%, 50% and 70% of vowel duration with confidence intervals for females and males COE of the two generations.

F1

Figure 5 :

 5 Figure 5: Median, lower and upper quartiles of the variable Ratio for females TAM.

Figure 6 :

 6 Figure 6: Median, lower and upper quartiles of the variable Ratio for males TAM.

  Likewise, this study reports 6 nasal vowels [ĩ], [ã], [õ], [ũ], [ɨ ]. However, no minimal pairs were found to demonstrate their contrastive properties. The minimal pairs given in previous studies show changes at the level of tone or glottalization that appear in certain contexts as evidenced by the examples c, d, e and, f.

Table 3 :

 3 Mean (and SE) F1, F2, F3 values (Hz) for vowel [ə], for females and males of the two generations and varieties TAM and COE.

10.21437/SIGUL.2023-6


Eloïse Equy

Jordana Hirtzel

Sophie Hellé

Béatrice Heurtault

Eric Mathieu

Morgane Rabineau

Vincent Ball
email: vball@unistra.fr

Lydie Ploux
email: ploux@unistra.fr

Fluorescent bioinspired albumin/polydopamine nanoparticles and their interactions with Escherichia coli cells

Keywords: accumulation, albumin, antibacterial, Escherichia coli, fluorescence, nanoparticles, penetration, polydopamine

Inspired by the eumelanin aggregates in human skin, polydopamine nanoparticles (PDA NPs) are promising nanovectors for biomedical applications, especially because of their biocompatibility. We synthesized and characterized fluorescent PDA NPs of 10-25 nm diameter based on a protein containing a lysine-glutamate diad (bovine serum albumin, BSA) and determined whether they can penetrate and accumulate in bacterial cells to serve as a marker or drug nanocarrier. Three fluorescent PDA NPs were designed to allow for tracking in three different wavelength ranges by oxidizing BSA/PDA NPs (Ox-BSA/PDA NPs) or labelling with fluorescein 5-isothiocyanate (FITC-BSA/PDA NPs) or rhodamine B isothiocyanate (RhBITC-BSA/PDA NPs). FITC-BSA/PDA NPs and RhBITC-BSA/PDA NPs penetrated and accumulated in both cell wall and inner compartments of Escherichia coli (E. coli) cells. The fluorescence signals were diffuse or displayed aggregate-like patterns with both labelled NPs and free dyes. RhBITC-BSA/PDA NPs led to the most intense fluorescence in cells. Penetration and accumulation of NPs was not accompanied by a bactericidal or inhibitory effect of growth as demonstrated with the Gram-negative E. coli species and confirmed with a Grampositive bacterial species (Staphylococcus aureus). Altogether, these results allow us to envisage the use of labelled BSA/PDA NPs to track bacteria and carry drugs in the core of bacterial cells.

Introduction

Organic nanoparticles (ONPs) are used to target and deliver drugs to tissue and eukaryotic cells [1,2], or bacteria and biofilms [3,4]. As nanovectors of drugs, they can deliver drugs locally, leading to a more efficient drug activity. Also, the required doses and the drug impact on healthy tissues compared to the free drug are lowered. Regarding the dramatic emer- [14]). (e) Possible locations of organic nanoparticles in bacterial cells. Figure 1c was adapted with permission from [13]. Copyright 2018 American Chemical Society. This content is not subject to CC BY 4.0.

gence and spreading of antimicrobial resistance of bacteria [START_REF] Van Dijk | Ad hoc advisory committee on disinfectants of the Health Council of the Netherlands[END_REF], this appears as a promising route to deliver antimicrobials while reducing the drug doses and subsequent harmful side effects in antibacterial applications. To this end, different types of ONPs have been used, such as liposomes [6] and nanoparticles (NPs) of poly(lactic-co-glycolic acid) (PLGA) [7], polycaprolactone [8], and chitosan [9]. Furthermore, fluorescent ONPs are a promising way to facilitate the localization of NPs in cells through fluorescence imaging. They can also be used for fluorescent labelling of cells, especially for live cell imaging, provided that the ONPs are harmless for cells. This has been developed for eukaryotic cells [10], but the use of ONPs for labelling bacterial cells is still rare and not described in literature for alive bacterial cells. The main limitation is probably the frequent cytotoxic effect of ONPs on bacteria.

Inspired by the eumelanin aggregates in human skin, polydopamine nanoaggregates (here referred to as nanoparticles, i.e., PDA NPs) have emerged as promising nanovectors for biomedical applications [11,12], especially because of their biocompatibility [13,14] and photothermic properties [15,16]. These properties can even be controlled by an external signal [17][18][19]. PDA NPs are formed upon oxidation in dopamine (DA) solutions with additives such as surfactants, polyelectrolytes, and proteins [14]. Eumelanin-like NPs with a diameter less than 20 nm have been obtained by this method. The additive plays a crucial role in the control of the NP size. Specifi-cally, Bergtold et al. demonstrated that a protein (e.g. chromofungin) containing a diad of lysine (K) and glutamate (E) (Figure 1a,b) in its sequence allows for the control of the formation of PDA NPs, in contrast to an additive without a KE diad (e.g., catestatin) [13]. During the formation process, hydroxy groups of dopamine form hydrogen bonds with carboxylic groups (COO -) of glutamate (pK a = 4.3), whereas protonated amino groups (NH 3 + ) of lysine (pK a = 10.5) further stabilize the aggregate by cation-π interactions with the aromatic ring of dopamine (Figure 1c). For this, K and E residues must be next to each other. Even a single glycine residue (G) located between K and E can destabilize the aggregates [13]. Among such possible additives, the albumin protein is an interesting candidate since it contains one KE diad and is already widely used in biology. Its hydrodynamic radius of about 4 nm at pH 7 makes it possible to envisage NP sizes close to 10 nm [20]. Chassepot and Ball prepared eumelanin-like particles in the presence of albumin, whose sizes decreased with the amount of protein down to 30 nm in diameter [14]. The structure of these albumin/ PDA NPs has not been elucidated completely. It has been demonstrated that proteins are present in the NPs' shell; they might also be present in the core (Figure 1d). Nevertheless, their potential both for fluorescent labelling of alive bacterial cells and as nanovector for antibacterial activity is high because of their small size and because any antibacterial natural or synthetic peptide containing KE diads may be used to create such PDA NPs. Fluorescent PDA NPs made with a KE diad-contain-ing protein or peptide have never been reported so far. They may be obtained by labelling with a fluorescent dye or oxidizing them, as reported by Ma et al. for other PDA NPs [21].

However, the capacity of PDA-based nanoparticles to target and penetrate bacteria for drug delivery, photothermic action, or fluorescent labelling of cells is unknown. Few publications have reported that nanoplastics can penetrate and accumulate in bacterial cells [22], thus suggesting that other ONPs may have a similar fate in bacteria. In general, the mechanisms of action of ONPs used as drug nanocarriers in antibacterial applications are expected to vary with the nanoparticle type (e.g., liposomes or PLGA NPs) but have not been elucidated so far. In contrast to inorganic NPs [23,24], it is unclear whether ONPs can penetrate bacterial cells. Alipour et al. have shown that a 170 nm diameter liposomal nanocarrier increased the accumulation of an antibiotic (polymyxin B) in Gram-negative bacterial cells, but the penetration of liposomes into the cell was not proved [25].

In general, organic nanocarriers are often reported to penetrate mammalian cells infected by bacteria, improving the drug accumulation in these eukaryotic cells and increasing the antibacterial efficiency of the drug [3,4,9,26]. However, the nanocarriers were not found in the bacterial cells, and the question was rarely mentioned at all. Thus, whether the increase in effectiveness of antibiotics carried by NPs is the result of the penetration of the complete nanocarrier-drug system into bacteria or rather an effect of the destabilization of the bacterial cell membrane by interactions with the nanocarriers (thus allowing for the penetration of the drug into the bacteria) is not known (Figure 1e). Yet, the accumulation of ONPs in bacterial cells is crucial if ONPs are to be used for fluorescent labelling of cells. Also, in the case of nanocarrier-drug systems, it may increase the dose of drug delivered close to the bacterial cell machinery and, therefore, improve the treatment efficacy. If the photothermic properties of the ONPs are to be exploited, the efficacy of the antibacterial treatment may also completely depend on the capacity of ONPs to penetrate and accumulate in the bacterial cell. Therefore, the fate of PDA NPs related to bacterial cells is a crucial aspect for their further use in antibacterial applications.

The objective of the study was to synthesize fluorescent PDA NPs based on a lysine-glutamate-diad containing protein and to determine whether they can enter and accumulate in bacterial cells. The investigation has been conducted with NPs made of polydopamine (PDA) and bovine serum albumin (BSA), and Escherichia coli (E. coli) bacteria as a bacterial model. Three different types of fluorescent BSA/PDA NPs have been designed to allow for the modification of their fluorescence properties. This also modified the outer surface chemistry; thus, the ability of the NPs to pass through the cell membrane was possibly facilitated. The localization of the fluorescent BSA/ PDA NPs related to the cells was investigated by high-resolution fluorescence imaging (high-resolution confocal microscopy). Also, the capacity of the pristine and fluorescent NPs to inhibit the growth of bacterial cells was evaluated through minimal inhibitory concentration (MIC) tests. The MIC value was also determined for Staphylococcus aureus (S. aureus).

Results and Discussion

Pristine BSA/PDA NPs with BSA/DA ratios ranging from 0. BSA/PDA NPs can be as small as 10 nm Synthesis of stable BSA/PDA NPs with size control

Pristine polydopamine nanoparticles (BSA/PDA NPs) were prepared according to Bergtold et al. [13] (Figure 2a-c). BSA and DA solutions were mixed in Tris buffer with ratios varying from 0.25 to 10. Contrary to a DA solution in Tris buffer, the mixtures of BSA and DA in Tris buffer inhibited the deposition of a PDA film on the wall of the reaction beakers (Figure 3a). This suggested that BSA/PDA aggregates formed and that almost all the free DA molecules were consumed in these aggregates, as already mentioned by Bergtold and co-workers [13].

A series of pristine BSA/PDA NPs with a mean diameter in number ranging from 66 ± 16 to 9 ± 3 nm was obtained (Figure 3b; see Supporting Information File 1, Table S1 for mean diameter in intensity) by increasing BSA/DA ratio from 0.25 to 10. The formation of BSA/PDA NPs is attributed to the interaction between DA and BSA, which contains the KE diad and allows for the control of the NP formation [13]. During the synthesis, DA is added into the BSA solution, thus avoiding that the proteins come into contact with already formed PDA. Bergtold et al. proposed that the size control is exerted by the specific interaction of the KE diad with DA [13]. Most of the DA molecules interact with the proteins, thus limiting the binding of proteins with each other or with PDA aggregates. This results in an increase in aggregate (i.e., particle) size. Hence, a rapid decrease in diameter was observed for BSA/DA ratios from 0.25 to 3. However, a plateau value was reached above a ratio of 6, leading to BSA/PDA NPs of 11 ± 3 nm and 9 ± 3 nm in diameter for BSA/DA ratios of 6 and 10, respectively. The size of the BSA protein is probably the limiting factor to a further decrease of the NP size. Indeed, one BSA molecule has an hydrodynamic diameter of 3-4 nm at physiological pH [20].

The resulting number N of nanoparticles per milliliter and selfpolymerization reaction yield η obtained with BSA/DA ratios of 3 and 10 were calculated with Equation 1 and Equation 2, respectively:

(

) 1
where V is the volume of the NP solution remainder after freeze-drying, m dry is the mass of the NP foam measured after freeze-drying, and ρ PDA is the density of polydopamine (1.52 g•cm -3 ) [27].

(

) 2
where C f is the final concentration after freeze-drying (m dry /V), is the initial concentration of dopamine (2 mg/mL), and is the initial concentration of BSA (6 mg/mL and 20 mg/mL for BSA/DA ratios of 3 and 10, respectively).

Final concentration and number of BSA/PDA NPs increased by increasing the BSA/DA ratio from 3 and 10 (2 × 10 14 and 4 × 10 15 NPs/mL, respectively), indicating a similar reaction yield of 85% (Supporting Information File 1, Table S2). The dispersion was changed by changing the ratio of BSA, but the reaction yield was maintained. This suggests that BSA acted as a "knife" changing the size, but not the composition, of the nanoparticles.

The suspension of BSA/PDA NPs was stable over two years of storage in Tris buffer in the dark at ambient temperature, as shown by the good dispersion and the absence of precipitates and deposition on the container wall (Figure 3c). Good stability and dispersion were maintained after dialysis with a 100 kDa cut-off membrane, which allowed for the removal of free BSA molecules. As shown by Chassepot and Ball [14] and Bergtold et al. [13], human serum albumin and other proteins play an important role in size control and stability of PDA NPs. Similarly, the stability of BSA/PDA NPs is probably due to BSA thanks to the strong PDA/KE interactions reported by Bergtold et al. This is supported by the progressive inhibition of the deposition of a PDA film on the wall of the reaction beaker, which was mentioned above and observed above a certain amount of BSA (Figure 3a). This allowed us to assume that BSA molecules below a critical amount cannot surround all DA molecules (some DA molecules thus form a thin film of PDA on the beaker's wall), whereas they stabilize PDA NPs above this amount.

The suspension stability was maintained when BSA/PDA NPs were in suspension in H 2 O or 150 mM NaCl solution. However, the mean hydrodynamic diameter varied with the solvent, with an increase by up to 50% for a BSA/DA ratio of 3 and 60% for a BSA/DA ratio of 10. The smallest BSA/PDA NPs had a mean diameter of 9 nm in 50 mM Tris and of 10 nm in H 2 O, but of 18 nm in 150 mM NaCl solution (Figure 3d). This increase was attributed to two co-existing phenomena related to the high salt concentration in 150 mM NaCl. First, the high ionic strength of 150 mM NaCl decreased long-range effect and intensity of electrostatic interactions compared to the other solvents, thus favoring other types of interactions (such as attractive van der Waals interactions) and, therefore, aggregation. Second, the increase in salt concentration might have led to an increase in the Stern layer thickness, resulting in a larger hydrodynamic diameter measured by DLS.

The size of BSA/PDA NPs remained stable as a function of pH at acidic pH, but increased moderately under alkaline conditions (Figure 3e). Chen et al. reported that PDA NPs deteriorated above pH 11 with, first, a decrease in size before the NP morphology changed into nanosheets [28]. However, in the case of BSA/PDA NPs, the NPs size increased when the pH value was above 6.5. In addition, precipitation, sedimentation, or deg-radation of the dispersion were not observed, showing that the suspension stability was unchanged. These results suggest that BSA prevented the degradation in alkaline solution, probably through heavily stabilizing the NPs.

The BSA/PDA NPs suspensions were freeze-dried to obtain dried NPs that could easily be stored and re-suspended at the needed concentration for further experiments. During the freeze-drying process, BSA/PDA NPs acted as surfactant and were located around water droplets when the water was rapidly sublimated, which resulted in the formation of a foam (Supporting Information File 1, Figure S1a). After freeze-drying, the foam could be easily re-suspended in Milli-Q ® water, resulting in a well-dispersed black suspension (Supporting Information File 1, Figure S1b). However, in the case of a BSA/DA ratio of 0.25, a powder was obtained instead of a foam (Supporting Information File 1, Figure S1c). This was probably due to a lack of BSA molecules to surround every molecule of PDA, which may have favored bridges involving BSA between NPs and further formation of aggregates (Supporting Information File 1, Figure S1d). This powder could not be re-dissolved in water, NaCl, or Tris buffer. Furthermore, the mean hydrodynamic diameter slightly increased for both BSA/DA ratios of 3 and 10 after dialysis and freeze-drying (22% and 18%, respectively) (Supporting Information File 1, Figure S1e). During water evaporation, NPs may have been brought closer, leading to the formation of irreversible interactions and slight aggregation. This only slight increase in size was considered insignificant regarding the possible effect on the interactions of NPs with bacteria; hence, freeze-drying was further used for the storage of BSA/PDA NPs.

Fluorescent modifications of BSA/PDA NPs

BSA/PDA NPs were modified to produce fluorescent BSA/ PDA NPs by three different methods, that is, the oxidation of pristine BSA/PDA NPs (Ox-BSA/PDA NPs) (Figure 2d) or the labelling of BSA with FITC or RhBITC fluorescent dyes (FITC-BSA/PDA NPs and RhBITC-BSA/PDA NPs, respectively) (Figure 2e). All fluorescent BSA/PDA NPs were made from pristine BSA/PDA NPs synthesized with a BSA/DA ratio of 10. This resulted in Ox-BSA/PDA NPs and FITC-BSA/PDA NPs with a mean diameter similar to that of pristine BSA/PDA NPs and RhBITC-BSA/PDA NPs of about 20 nm in diameter (Figure 3f). A small decrease in size of Ox-BSA/PDA NPs was expected and measured (30% decrease), as also noticed by Ma et al., probably due to the degradation of the pristine BSA/PDA NPs upon oxidation [21]. However, regarding the size values and deviations measured, the decrease was considered insignificant here. The increase in size of RhBITC-BSA/PDA NPs upon labelling with RhBITC can be explained by the simultaneous presence of a positive charge and two additional diethylamino groups in RhBITC, which are absent in FITC. These two additional chemical moieties may have induced some weak aggregation between the negatively charged RhBITC-BSA/PDA NPs, in contrast to FITC-BSA/PDA NPs.

Ox-BSA/PDA NPs were produced by the oxidation of BSA/ PDA NPs for 24 h with H 2 O 2 . The BSA/PDA NP suspension, initially dark brown, turned to translucent light brown during oxidation (Supporting Information File 1, Figure S2b). Under UV light, the Ox-BSA/PDA NPs suspension emitted fluorescence, in contrast to the suspension before oxidation (Supporting Information File 1, Figure S2c). This may result from the conjugation and electronic density changes induced during the reaction with H 2 O 2 , during which hydroxy groups were oxidized into ketones. Eventually, the large majority of polydopamine functional groups are expected to be ketones with only a few remaining of hydroxy groups (Supporting Information File 1, Figure S2a). In addition, labelling of pristine BSA/ PDA NPs with FITC and RhBITC dyes led to an obvious change in color of the suspension, from brown (Supporting Information File 1, Figure S3a) to yellow and red (Supporting Information File 1, Figure S3b,c), respectively, in agreement with the color of the corresponding dye.

Pristine BSA/PDA-NPs exhibited a wide absorption spectrum with a maximum of absorption at 280 nm, whereas Ox-PDA/ BSA-NPs showed a less broad and intense absorption with a maximum at 285 nm (Figure 4a). In other words, the oxidation leads to a bathochromic (increase in wavelength) shift due to an increase in conjugation when -OH groups were oxidized into =O groups. Moreover, a hypochromic (decrease in absorption intensity) effect was measured, which may have resulted from the degradation of the BSA/PDA-NPs upon oxidation (suggested by the insignificant decrease in size of Ox-BSA/PDA NPs compared to pristine BSA/PDA NPs) and a reduction of concentration or molar extinction coefficient during the prolonged oxidation. Indeed, it is well known that the oxidation of indole quinone groups in melanin-like materials leads to the release of pyrrole carboxylic acids [29]. In addition, Ox-BSA/ PDA NPs revealed an emission spectrum centered around 490 nm. It is to note that no significant shift of the maximum emission wavelength depending on the excitation wavelength was measured (Supporting Information File 1, Figure S4a), contrary to what has been mentioned in the literature [21,30]. This effect is not elucidated so far, but it is important to note that the NPs considered here are different from those of Zhang and Ma, since PDA is here associated to BSA. Moreover, the Stokes shift of Ox-BSA/PDA NPs (λ max ems -λ max abs ) is estimated to be about 200 nm (Supporting Information File 1, Figure S4b), which is higher than that of common fluorochromes such as RhBITC and FITC (about 25 nm) [31] or even DAPI (about 100 nm) [32]. This big difference in energy is consistent with the ability of PDA to heat up under irradiation by non-radiative relaxation. This is favorable to minimize the superposition between absorption and emission peaks, but might lead to a low quantum yield and, thus, poor emission intensity. Indeed, the quantum yield of Ox-BSA/PDA NPs in water (Φ) was calculated to be 0.1% with Equation 3, using DAPI in water as the reference [33].

(3) where, Φ DAPI is the fluorescence quantum yield of DAPI when unbound to DNA (4%) [32],

and are the integrated fluorescence intensities of the light emitted by Ox-BSA/PDA NPs and DAPI, respectively, A Ox-BSA/PDA NPs and A DAPI are the absorption values of Ox-BSA/PDA NPs and DAPI solutions, respectively, and η Ox-BSA/PDA NPs and η DAPI are the refractive indices of the solvents of Ox-BSA/PDA NPs and DAPI solutions, respectively. Because the solvent is the same (water) for both Ox-BSA/PDA NPs and DAPI solutions, the value of is here equal to 1.

Compared to the quantum yields of FITC, RhBITC, and DAPI bound to DNA, which are in the range of 40-90% at room temperature [31,32,34], Ox-BSA/PDA NPs reveal low emission efficiency. However, even if the quantum yield of Ox-BSA/ PDA NPs is low, it is significantly higher than the quantum yield of non-oxidized NPs, which are not fluorescent at all [35].

FITC-and RhBITC-BSA/PDA NPs exhibited absorption maxima at 500 and 560 nm, respectively, shifted by 8 nm compared with free FITC and RhBITC (absorption maxima at 492 and 552 nm, respectively) (Figure 4c,d and Supporting Informa-tion File 1, Figure S5). This probably results from a change in polarity in the close environment of the bound fluorophore. In addition, the absorption intensity of FITC-BSA/PDA and RhBITC-BSA/PDA NPs above 600 nm is a typical feature of melanin materials. The high background (decreasing from 400 to 800 nm and similar to the absorption spectrum of pristine BSA/PDA NPs) was attributed to the absorption by NPs (Figure 4a). The emission spectra of FITC-and RhBITC-BSA/ PDA NPs were centered around 520 and 578 nm, respectively, which is similar to free FITC and RhBITC (emission maxima at 519 and 578 nm, respectively) under excitation at 488 and 550 nm, respectively (Figure 4c,d and Supporting Information File 1, Figure S5). Unfortunately, because PDA absorbs a significant part of the light emitted by the fluorophores, the intensity cannot be related to the number of FITC-and RhBITClabelled NPs. However, the absorption intensities were used to estimate (even though roughly) the equivalent quantities of free FITC and RhBITC to be used as controls in the further bacteriological assays. The estimated equivalent quantities corresponding to a concentration of fluorescent NPs of 2 mg/mL (164 µM for FITC and 373 µM for RhBITC) were calculated as described in the Experimental section.

The stability of the fluorescent labelling was tested after four months of ageing of FITC-and RhBITC-BSA/PDA NPs. Dialysis was performed on the four months old NP suspensions to retrieve FITC or RhBITC molecules possibly released from the labelled NPs by following the same procedure as described below for the elimination of unbound labelling molecules (see Experimental section). The fraction of released dye was estimated on the basis of the emission intensity of the dialysate and the initial quantity of labelled NPs (Supporting Information File 1, Figure S6). The fractions were less than 1% (1.5 µM for FITC and 0.5 µM of RhBITC) of the initial dye quantity, showing the high stability of the labelling and of the labelled NPs.

FITC-BSA/PDA NPs and RhBITC-BSA/PDA NPs tend to accumulate in E. coli cells with heterogeneous patterns

Whether pristine and fluorescent BSA/PDA NPs can interact with bacterial cells (especially E. coli) is unknown. Therefore, the possible accumulation of pristine and fluorescent BSA/PDA NPs on and in cells was studied with the smallest NPs (about 10 nm diameter) since NP penetration in cells is expected to be inversely related to NP size [36].

The accumulation of pristine and fluorescent BSA/PDA NPs was evaluated by standard and high-resolution fluorescence confocal microscopy after 24 h of contact of NPs with E. coli cells. Obviously, bacteria without NPs and bacteria with pris-tine BSA/PDA NPs were not detected at excitation and emission wavelengths corresponding to FITC and RhBITC dyes. It was also impossible to identify the specific fluorescent signal emitted by Ox-BSA/PDA NPs in absence of an adequate laser for the excitation of Ox-BSA/PDA NPs at 285 nm. Under excitation with a higher wavelength (405 nm), a low fluorescence signal was detected in the 460-541 nm and 415-482 nm ranges with the standard and the high-resolution microscope, respectively (Supporting Information File 1, Figure S7a). In part, it may result from the fluorescence of Ox-BSA/PDA NPs at λ exc = 405 nm; however, it is more probably due to the intrinsic fluorescence of bacteria. Indeed, E. coli cells alone and E. coli cells cultivated with free FITC or RhBITC, or FITC-or RhBITC-BSA/PDA NPs revealed a similar fluorescence signal when excited at 405 nm (Supporting Information File 1, Figure S7a). This can be attributed to the intrinsic fluorescence of bacteria under 405 nm excitation due to porphyrins [37,38]. With Ox-BSA/PDA NPs, no significant difference in fluorescence intensity could be detected in this emission range under 405 nm excitation compared to the intrinsic fluorescence background. This prevents a conclusion on whether Ox-BSA/PDA NPs may have accumulated on or in E. coli cells.

Under excitation at 488 nm, cells cultivated with FITC-BSA/ PDA NPs and free FITC emitted slight fluorescence the 495-634 nm and 496-565 nm ranges (observed with standard and high-resolution microscopes, respectively) (Figure 5a, Supporting Information File 1, Figure S7c), in contrast to E. coli cells alone or cultivated with RhBITC-BSA/PDA NPs or free RhBITC. Under excitation at 561 nm, E. coli cells cultivated with RhBITC-BSA/PDA NPs or free RhBITC emitted fluorescence in the 567-703 nm and 553-628 nm ranges (observed with standard and high-resolution microscopes, respectively) (Figure 5a, Supporting Information File 1, Figure S7). These emission ranges are in agreement with the fluorescence characteristics of FITC-BSA/PDA NPs and RhBITC-BSA/PDA NPs (Figure 4c,d) and can, thus, be attributed to FITC or RhBITC. Hence, FITC alone, RhBITC alone, FITC-BSA/PDA NPs, and RhBITC-BSA/PDA NPs were all able to label E. coli cells. However, a much higher intensity was observed with RhBITC and RhBITC-BSA/PDA NPs than in the other experiments as confirmed by quantifying the maximal fluorescence measured in the micrographs (Figure 5b). This may result from the positive charges carried by RhBITC at physiological pH, which are absent in the FITC molecules. The positive charges are expected to participate in attractive electrostatic interactions with the negatively charged bacterial membrane, that is, the phosphate groups of their phospholipids. This facilitates the intimate contact between RhBITC-BSA/PDA NPs and the bacterial membrane and, possibly, the subsequent penetration of the NPs.

The localization of the fluorescence emission related to bacterial cells was determined on the basis of micrographs extracted from 3D-stack images obtained with the high-resolution confocal laser scanning microscope (CLSM, Stellaris 5, Leica Biosystems, Wetzlar, Germany). In all experiments with dyes (FITC-BSA/PDA NPs, RhBITC-BSA/PDA NPs, free FITC, and free RhBITC in equivalent concentrations), the fluorescence signal related to RhBITC or FITC was emitted at the location of the cells (Figure 5a,c and Figure 6). The fluorescence intensity in cells incubated with RhBITC-BSA/PDA NPs was about 15% of the intensity in cells incubated with the equivalent quantity of free RhBITC (Figure 5b). It reached about 100% with FITC-BSA/PDA NPs compared to free FITC. These ratios are much higher than expected if only free dye molecules released from NPs had penetrated the cells (1% as estimated above). Hence, they prove that the fluorescence measured in cells was not emitted by free molecules of dye but by molecules bound to NPs.

More specifically, the fluorescence appeared to come from the cell wall and from some inner parts of the bacterial cells with a frequent location at the poles of the cell. The fluorescence profiles confirm (i) that dyes accumulate in the cell wall rather than on the outside as shown by the co-localization of the intrinsic (blue) and dye-related fluorescence signals (dashed lines and black arrows in Figure 6b, Supporting Information File 1, Figure S8) and (ii) that the fluorescence was present as clusters in inner cell compartments (empty arrows in Figure 6b, Supporting Information File 1, Figure S8). This accumulation of labelled BSA/PDA NPs may be related to proteins since fluorescent dyes such as RhBITC can interact with proteins in a non-covalent manner [39]. In addition, proteins can aggregate at the poles or in the cytosol of E. coli (and other bacterial species) during cell division or as the result of cellular ageing or under external stressors [40,41]. This may match the distribution patterns observed with FITC-and RhBITC-BSA/PDA NPs. Moreover, by themselves or by the excitation light used to reveal them, the labelled NPs may be the external stressor leading to cell ageing and subsequent protein aggregation as proposed by Rang and co-workers [42]. In any case, our results indicate that FITC-and RhBITC-BSA/PDA NPs were able to penetrate and accumulate in the cell wall and internal compartments of E. coli cells in significant quantities.

BSA/PDA NPs are prone to damage bacterial cells since they can penetrate them. The possible harmful impact on bacterial growth was therefore tested with E. coli and S. aureus. However, we showed that BSA/PDA NPs in concentrations corresponding to more than 10 7 NPs per bacterial cell (0.2-2.0 mg/mL; 4 × 10 13 to 4 × 10 14 NPs/mL) were unable to significantly modify the growth of a E. coli population (Figure 6a). This was maintained regardless of the surface properties of the NPs since pristine, Ox-, and RhBITC-BSA/PDA NPs all failed to inhibit E. coli growth (Figure 7b). It can be noted that the population growth was even slightly but significantly favored in the presence of pristine and fluorescent BSA/ PDA NPs. This effect, which is not elucidated so far, was also observed regarding the growth of S. aureus populations (Supporting Information File 1, Figure S9). Especially the absence of E. coli growth inhibition by RhBITC-BSA/PDA NPs allows us to envisage the use of the labelled BSA/PDA NPs developed in this study to track bacterial cells, but also to carry drugs in bacterial cells thanks to their penetration capacity. allows us to envisage fluorescent tracking of live bacterial cells using these fluorescent NPs. The capacity of these NPs to penetrate cell walls might also enable their use to carry drugs in the core of bacterial cells.

Experimental Synthesis

Reagents Dopamine (DA) hydrochloride and bovine serum albumin (BSA) were purchased from Merck (Darmstadt, Germany) and used without purification. Fluorescein isothiocyanate (FITC), rhodamine B isothiocyanate (RhBITC), hydrochloric acid 37% (wt/v), absolute ethanol and Hellmanex ® were purchased from Merck (Darmstadt, Germany). Tris(hydroxymethyl)aminomethane (Tris buffer) was purchased from Euromedex (Strasbourg, France). 4′,6-Diamidino-2-phenylindole dihydrochloride (DAPI) was purchased from Fisher Scientific (Illkirch, France). All aqueous solutions were made using Milli-Q ® water (ρ = 18.2 MΩ•cm; Millipore ® Reverse Osmosis system; Merck, Darmstadt, Germany).

BSA/polydopamine nanoparticles

Polydopamine nanoparticles (BSA/PDA NPs) were prepared according to Bergtold and co-workers [13] (Figure 2a-c).

Tris buffer solution was prepared by dissolution of 6.055 g (5 × 10 -2 mol) of Tris powder in 1 L of Milli-Q ® water. The pH was adjusted to 8.5 by adding hydrochloric acid. X mg of BSA (X depending on the chosen BSA/DA wt/wt ratio; see Table 1) were slowly dissolved in 100 mL Tris buffer to avoid foam formation. 200 mg of dopamine hydrochloride was added to yield a solution of 2 mg/mL of DA. This solution was gently shaken at ambient temperature in the dark. After 24 h, a homogenous black suspension of BSA/PDA NPs was obtained. Small oligomers, unreacted dopamine monomers, and Tris salt were removed from the reaction medium by dialysis. 20 mL of the suspension were dialyzed against 2 L of Milli-Q ® water using a Spectra/Por ® dialysis membrane with a molecular weight cutoff of 8-10 kDa (purchased from Fisher Scientific S.A.S., Illkirch, France). The dialysis was performed in the dark and followed by spectroscopic measurements at 280 nm (absorption of dopamine) to check for the absence of oligomers in the dialysate at the end of the dialysis process [13]. Dialysis was repeated three times, yielding a total dilution of the dialysate by a factor of 10 6 . The solution was frozen at -80 °C for 1 h, and water was eliminated by freeze-drying (Alpha 1-4 LD plus ; Christ, Osterode am Harz, Germany) for 48 h to provide lyophilized foam, which was stored at room temperature and protected from light before use. Fluorescent BSA/polydopamine nanoparticles Fluorescent BSA/PDA nanoparticles with a BSA/DA ratio of 10 were synthesized by using two different ways: (i) by oxidation of pristine BSA/PDA nanoparticles and (ii) by adding fluorescent labels to the BSA used to prepare BSA/PDA nanoparticles (Figure 2d-e).

PDA oxidation. Fluorescent, oxidized polydopamine nanoparticles (Ox-PDA/BSA NPs) were prepared on the basis of pristine BSA/PDA NPs synthesized as described above with a BSA/DA ratio of 10 (1.1 × 10 -6 mol BSA, 1 equiv). Pristine BSA/PDA NPs were then modified by a procedure inspired from Ma et al. [21] as follows: A suspension of BSA/PDA NPs foam was prepared in Milli-Q ® water at a concentration of 10 mg BSA/PDA NPs per milliliter; 7.5 mL of this suspension (pH 6.8) and 2 mL of H 2 O 2 solution (30 wt %) were stirred at room temperature in the dark for 24 h. Residual H 2 O 2 was removed from the reaction medium by dialyzing about 8 mL of the suspension against 1 L Milli-Q ® water using a Spectra/Por ® dialysis membrane with a molecular weight cut-off at 8-10 kDa. The dialysis was done in the dark and repeated three times yielding a total dilution of the dialysate by a factor of 10 6 .

Labelling of BSA/PDA NPs with a fluorescent probe. Fluorescently labelled polydopamine nanoparticles (FITC-and RhBITC-PDA/BSA NPs) were prepared on the basis of pristine BSA/PDA NPs synthesized as described above with a BSA/DA ratio of 10 (1.1 × 10 -6 mol BSA, 1 equiv). Pristine BSA/PDA NPs were then modified by labelling BSA with the commercial fluorescent probes FITC and RhBITC to obtain FITC-BSA/ PDA NPs and RhBITC-BSA/PDA NPs, respectively. BSA and the fluorescent probes were linked through the isothiocyanate groups of FITC and RhBITC and the amine group of BSA. To prepare FITC-BSA/PDA NPs, 80 mg of dehydrated foam of pristine BSA/PDA NPs were dissolved into 8 mL of sodium carbonate buffer (50 mM, pH 8.5). A concentrated solution of FITC (8.25 mM) was prepared by dissolving 3.2 mg into 1 mL of DMSO. 400 μL of this solution was added to the pristine BSA/PDA NPs solution to reach a FITC concentration of 4 × 10 -4 M (3.3 × 10 -6 mol, 3 equiv). The reaction was performed in the dark for 1 h and followed by a dialysis against 1 L of Milli-Q ® water using a Spectra/Por ® dialysis membrane with a molecular weight cut-off at 8-10 kDa to eliminate unbound labelling molecules. The dialysis was done in the dark and repeated three times yielding a total dilution of the dialysate by a factor of 10 6 . The evolution of the dialysis was followed by measuring the absorption spectrum of the dialysate. The same procedure was used to prepare RhBITC-BSA/PDA NPs, except that FITC was replaced by RhBITC. The concentrated solution of RhBITC (8.25 mM) was prepared by dissolving 4.4 mg in 1 mL of DMSO.

Characterization of the BSA/polydopamine nanoparticles Size measurement

The diameter of the NPs was measured by dynamic light scattering (DLS) using a Zetasizer Nano ZS from Malvern Panalytical (Malvern, UK). Measurements were performed while taking into account a refractive index of 1.73 -0.02i for PDA (the imaginary part corresponds to the absorption coefficient) at a wavelength of 589 nm, that is, close to the wavelength of the laser used in the device (633 nm). Samples were diluted to get an absorption below 0.1. Size distribution results are given in intensity and can be expressed also in volume or number. Standard deviations of the sample's mean hydrodynamic diameters as well as the polydispersity index are provided in Supporting Information File 1, Table S1. In this study, results given in number will be used.

Characterization of the fluorescence properties

Evaluation of the antibacterial activity

The antibacterial activity of pristine NPs, Ox-, and RhBITC-BSA/PDA NPs with a BSA/DA ratio of 10 was determined by calculating the bacterial growth inhibition from absorption measurements at 620 nm. Measurements were acquired with a Multiskan spectrophotometer (Fisher Scientific, Illkich, France). One colony of E. coli or S. aureus was transferred from the agar plate into fresh liquid LB (for E. coli) or MH (for S. aureus) and incubated at 37 °C overnight. The bacterial suspension was then diluted with fresh LB (for E. coli) or MH (for S. aureus) to 6 × 10 6 CFU/mL. Lyophilized foam of BSA/ PDA NPs with a BSA/DA ratio of 10 was diluted with 9 mg/mL NaCl solution to obtain a 20 mg/mL suspension of BSA/PDA NPs. This suspension was filtered in a microbiological safety cabinet with a 0.20 µm filter to eliminate possible contaminations and diluted in 9 mg/mL NaCl solution to prepare suspensions with NP concentrations of 0.5, 1, 2, 5, and 10 mg/mL. Finally, 10 µL of BSA/PDA NP suspension was added to 90 µL of bacterial solution (either E. coli or S. aureus) placed in 96 well plates to prepare bacterial suspensions with concentrations of BSA/PDA NPs from 0 to 2000 µg/mL (0 µg/mL (negative control C-), 50 µg/mL, 100 µg/mL, 200 µg/mL, 500 µg/mL, 1000 µg/mL, and 2000 µg/mL). A 10 µL mixture of tetracycline (10 µg/mL) and cefotaxime (0.1 µg/mL) was used as a positive control (C+) of bacterial growth inhibition. Bacterial growth was assessed from the absorption at 620 nm after 24 h of incubation at 37 °C. Each assay was performed three times.

The statistical significance of two-by-two differences between the mean absorption at 620 nm was determined by unilateral Student's tests. Bacterial growth inhibition was calculated for each concentration of NPs via Equation 4.

(

) 4
where A bact+NPs , A NPs , A bact , and A LB are the absorption values at 620 nm of the bacterial suspension containing NPs at a given concentration (A bact+NPs ), the bacteria-free suspension containing NPs at a given concentration (A NPs ), the bacterial suspension without NPs (A bact ), and the suspension with neither bacteria nor NPs (A LB ), respectively.

The minimal inhibitory concentration (MIC) corresponds to the lowest concentration of NPs that significantly inhibits growth of bacteria. If no inhibition effect is observed in the range of concentration tested, then the MIC will be indicated as higher than the maximum concentration tested.

Determination of the localization of NPs in bacterial cells

Standard and high-resolution fluorescence confocal laser scanning microscopy (CLSM) were used to determine the localization of the fluorescent BSA/PDA NPs with a BSA/DA ratio of 10 in bacterial cells. One colony of E. coli was transferred from the agar plate into fresh liquid LB and incubated at 37 °C overnight. The bacterial suspension was then diluted with LB to obtain a transmittance at 600 nm of 0.001 (6 × 10 6 CFU/mL). 200 µL of fluorescent Ox-, FITC-, or RhBITC-BSA/PDA NPs suspension was added to 800 µL of the bacterial suspension, corresponding to the highest concentration of NPs tested in the previous assay, that is, 2 mg/mL of NPs. Three controls were also prepared: one with bacteria alone, one with NPs alone, and one with bacteria, without NPs but with fluorescent dye, either FITC or RhBITC, in a concentration equivalent to the quantity used to label the NPs. Because PDA absorbed a significant part of the light emitted by the fluorophores, the emission could only be roughly estimated by the difference between the intensity of the dye solution added to label BSA/PDA NPs and the intensity of the dialysis water. The equivalent dye concentrations (corre- sponding to 2 mg/mL of fluorescent NPs) were thus estimated to be 164 and 373 µM for FITC and RhBITC, respectively. The suspensions were all incubated at 37 °C for 24 h. Then, they were diluted in fresh liquid LB to reach a transmittance of 0.5 at 600 nm for suspensions without NPs, or a transmittance of 0.5 increased by the transmittance of the control with NPs alone, for samples containing NPs (to take into account the transmittance due to NPs). Afterward, samples were centrifuged at 3000 rpm for 10 min and re-suspended in 1 mL of PBS. This procedure was repeated two times. To allow for the storage of the samples over days, a fixation step was added to the preparation procedure. However, it is important to specify that this step is not required and that bacteria stained by the RhBITC-or FITC-BSA/PDA NPs can be observed alive directly after staining. The samples were fixed with paraformaldehyde (PFA): Samples were centrifuged at 3000 rpm for 10 min, re-suspended in 500 µL PBS 2× and 500 µL of PFA 8%, then centrifuged again at 3000 rpm for 10 min and re-suspended and incubated for 1 h in PFA 4% in PBS 1×. Samples were washed by one last cycle of centrifugation (3000 rpm, 10 min) and re-suspension in PBS (1 mL). 8 µL of the suspension was finally deposited on a glass slide and covered by a glass coverslip, both previously washed using 0.2% Hellmanex ® cleaning solution and ethanol in an ultrasonic bath. After complete drying (15 min), 8 µL of mounting medium (ProLong™ Diamond, Molecular Probes™) was deposited on the dry drop of sample, before adding a cover glass and sealing it with nail polish. The samples were kept at 4 °C in the darkness.

Samples were observed with a standard CLSM Zeiss LSM710 (Zeiss, Oberkochen, Germany) equipped with a 63× objective lens (X63 PL APO oil/1.4) and a high-resolution confocal microscope Stellaris 5 (Leica Biosystems, Wetzlar, Germany) equipped with a 100× objective lens (X100 HCX PL APO oil/ 1.4). Using the LSM710 microscope, excitation and emission wavelengths were 405, 488, and 561 nm, and in the ranges of 460-541, 495-634, and 567-703 nm, respectively. Using the Stellaris microscope, excitation and emission wavelengths were 405, 488, and 543 nm, and in the ranges of 415-482, 496-565, and 553-628 nm, respectively. With the Stellaris microscope, 3D-stack images with thicknesses from 1.5 to 5.0 µm with steps from 125 to 145 nm were recorded to allow for the localization of fluorescence emission related to the bacterial cells. Eight different types of samples were prepared, which are described in Table 2.

Figure 1 :

 1 Figure 1: Molecules of (a) lysine (K) and (b) glutamate (E). (c) Interactions between dopamine and KE sequence. (d) Possible structures of protein (grey)/polydopamine (black) composite particles (inspired from[14]). (e) Possible locations of organic nanoparticles in bacterial cells. Figure1cwas adapted with permission from[13]. Copyright 2018 American Chemical Society. This content is not subject to CC BY 4.0.

Figure 2 :

 2 Figure 2: (a) Photographic image of a dopamine solution. Main reactions leading to an auto-polymerization of dopamine to (b) polydopamine or (c) to polydopamine nanoparticles in the presence of BSA (inspired from [13]). (d) Synthesis principle and photographic images of suspensions of fluorescent Ox-BSA/PDA NPs obtained by oxidation of pristine BSA/PDA NPs. (e) Synthesis principles and photographic images of fluorescent RhBITC-BSA/PDA NPs and FITC-BSA/PDA NPs obtained by using RhBITC-and FITC-labelled BSA, respectively.

Figure 3 :

 3 Figure 3: Solutions and sizes of pristine and fluorescent BSA/PDA NPs. (a) Evidence of the inhibition of a PDA film deposition on the wall of the reaction beaker of (left) BSA/PDA solution in Tris buffer compared to (right) PDA solution in Tris buffer. (b) Mean hydrodynamic diameter of pristine BSA/ PDA NPs as a function of the BSA/DA ratio. (c) Photograph of pristine BSA/PDA NPs after two years of storage. (d) Mean hydrodynamic diameter in number of pristine PDA/BSA-NPs depending on the solvent. (e) Mean hydrodynamic diameter in number of pristine PDA/BSA-NPs depending on the pH value. (f) Mean hydrodynamic diameter in number of pristine BSA/PDA NPs, Ox-BSA/PDA NPs, FITC-BSA/PDA NPs, and RhBITC-BSA/PDA NPs (all synthesized with a BSA/DA ratio of 10).

Figure 4 :

 4 Figure 4: Absorption and emission spectra of pristine BSA/PDA NPs, Ox-BSA/PDA NPs, FITC-BSA/PDA NPs, and RhBITC-BSA/PDA NPs. (a) Absorption of pristine BSA/PDA NPs before and after oxidation. (b) Normalized absorption and fluorescence emission (excitation wavelength λ exc = 375 nm) spectra of Ox-BSA/PDA NPs. (c) Normalized absorption and fluorescence emission (excitation wavelength λ exc = 488 nm) spectra of FITC-BSA/PDA NPs. (d) Normalized absorption and fluorescence emission (excitation wavelength λ exc = 550 nm) spectra of RhBITC-BSA/PDA NPs. Spectra were smoothed using the moving average method on three points.

Figure 5 :

 5 Figure 5: Fluorescence characterization of E. coli cultures with FITC-BSA/PDA NPs, free FITC, RhBITC-BSA/PDA NPs, free RhBITC, or alone (NPs synthesized with a BSA/DA ratio of 10). (a) Typical fluorescence micrographs extracted from 3D-stack images measured with a high-resolution CLSM (Stellaris 5, Leica Biosystems, Wetzlar, Germany) (λ exc of 405, 488, or 543 nm; range of λ em of 415-482, 496-565, and 553-628 nm, respectively). (b) Maximal fluorescence intensity measured in bacterial cells for each condition (mean ± SD of all the micrographs of each condition). (c) Mean fluorescence intensity of bacterial cells for each condition (mean counts in bacterial cells per field) (mean ± SD of all complete bacteria in all micrographs of each condition).

Figure 6 :

 6 Figure 6: Localization of the fluorescence emission related to E. coli cells. (a) Orthogonal views extracted from a 3D stack of a RhBITC-BSA/PDA NPs with E. coli sample measured with a high-resolution CLSM (Stellaris 5, Leica Biosystems, Wetzlar, Germany) (λ exc of 405 and 543 nm; range of λ em of 415-482 and 553-628 nm, respectively). (b) Longitudinal and transversal profiles of fluorescence intensity of a typical bacterial cell cultivated with RhBITC-BSA/PDA NPs (profiles are identified with numbers as displayed on the micrograph measured with the high-resolution Stellaris 5 CLSM as described in (a)).

Figure 7 :

 7 Figure 7: Growth inhibition of E. coli populations with pristine BSA/PDA NPs from 0.2 to 2 mg/mL concentration or with antibiotics (solution of 10 µg/mL tetracycline and 0.1 µg/mL cefotaxime) ("T+C"; positive control) compared to E. coli culture without NPs (a), and with pristine and fluorescent Ox-BSA/PDA NPs and RhBITC-BSA/PDA NPs (1 mg/mL concentration) compared to E. coli culture without NPs (b). * and ** indicate significant differences to E. coli culture alone (p < 0.01 and p < 0.001, respectively).

  25 to 10 and three different types of fluorescent BSA/PDA NPs with a BSA/DA ratio of 10 were synthesized and characterized. Three synthesis routes were used to prepare these fluorescent BSA/PDA NPs, based either on the oxidation of pristine BSA/

PDA NPs (Ox-BSA/PDA NPs) or on labelling with fluorescein 5-isothiocyanate (FITC) or rhodamine B isothiocyanate (RhBITC) fluorescent dyes (FITC-BSA/PDA NPs and RhBITC-BSA/PDA NPs, respectively). According to Ma et al., Ox-BSA/PDA NPs are expected to emit a maximum of fluorescence in the 450-500 nm range under 405 nm light irradiation

[21]

. FITC-BSA/PDA NPs and RhBITC-BSA/PDA NPs are expected to emit similarly to free FITC (emission in the green range if excited at 488 nm) and RhBITC (emission in the red range if excited at 561 nm), respectively.

Table 1 :

 1 Weight of BSA (X mg) and dopamine (DA), and the corresponding BSA/DA ratio used to synthesize the BSA/PDA NPs.

	BSA/DA ratio	BSA weight (mg)	DA weight (mg)
	0	0	200
	0.25	50	200
	1.00	200	200
	3.00	600	200
	6.00	1 200	200
	10.00	2 000	200

Table 2 :

 2 Samples prepared for fluorescence confocal microscopy.

	Sample composition	E. coli concentration (CFU/mL) NP concentration (mg/mL) Fluorescent dye concentration
				(in NPs or free) (µM)
	BSA/PDA NPs + E. coli	6 × 10 6	2	0
	Ox-BSA/PDA NPs + E. coli	6 × 10 6	2	0
	FITC-BSA/PDA NPs + E. coli	6 × 10 6	2	164 a
	RhBITC-BSA/PDA NPs + E. coli	6 × 10 6	2	373 a
	BSA/PDA NPs	0	2	0
	Ox-BSA/PDA NPs	0	2	0
	FITC-BSA/PDA NPs	0	2	164 a
	RhBITC-BSA/PDA NPs	0	2	373 a
	FITC + E. coli	6 × 10 6	0	164
	RhBITC + E. coli	6 × 10 6	0	373
	E. coli	6 × 10 6	0	0

a Estimation based on the difference in absorption intensity of the fluorescent dye solution used to label BSA/PDA NPs and the absorption intensity of the dialysis water.

Supporting Information

Supporting Information File 1 provides additional tables and figures regarding synthesis, characteristics, and properties (including accumulation in bacterial cells) of pristine and fluorescent BSA/PDA NPs.

Supporting Information File 1

Additional tables and figures.

[https://www.beilstein-journals.org/bjnano/content/ supplementary/2190-4286-14-100-S1.pdf]

Funding

The authors would like to thank Region Grand Est (ERMES project), ANR (Agence Nationale de la Recherche) (project No. ANR-21-CE17-0020-0) and the "Groupement de Recherche" GDR B2I (GDR3751) for their support.

ORCID ® iDs

Morgane Rabineau -https://orcid.org/0000-0001-7325-7518 Vincent Ball -https://orcid.org/0000-0002-7432-4222 Lydie Ploux -https://orcid.org/0000-0003-0410-693X

License and Terms

This is an open access article licensed under the terms of the Beilstein-Institut Open Access License Agreement (https://www.beilstein-journals.org/bjnano/terms), which is identical to the Creative Commons Attribution 4.0 International License (https://creativecommons.org/licenses/by/4.0). The reuse of material under this license requires that the author(s), source and license are credited. Third-party material in this article could be subject to other licenses (typically indicated in the credit line), and in this case, users are required to obtain permission from the license holder to reuse the material.

The definitive version of this article is the electronic one which can be found at: https://doi.org/10.3762/bjnano.14.100