Hannah M Ter Hofstede
email: hannah.ter.hofstede@dartmouth.edu

Stefan Schöneich

Tony Robillard

Berthold Hedwig

Evolution of a communication system by sensory exploitation of startle behavior

. In one group of crickets (Eneopterinae: Lebinthini), however, males produce exceptionally high-frequency calling songs in the range of bat calls [8], a surprising signal in the context of mate attraction. We found that female lebinthines, instead of approaching singing males, produce vibrational responses after male calls, and males track the source of vibrations to find females. We also demonstrate that field cricket species closely related to the Lebinthini show an acoustic startle response to high-frequency sounds that generates substrate vibrations similar to those produced by female lebinthine crickets.

. In lebinthine crickets, however, we found that auditory ascending neurons are only tuned to high-frequency sounds, and their tuning matches the thresholds for female vibrational signals. Our results demonstrate how sensory exploitation of antipredator behavior can evolve into a communication system that benefits both senders and receivers.

RESULTS

Animals generate signals using a variety of modalities to influence the behavior of receivers, and various mechanisms have been proposed for the evolution of these diverse signals [START_REF] Bradbury | Principles of Animal Communication[END_REF]. In some cases, novel signals evolve because they tap into a pre-existing sensory bias and associated behavioral response in the receiver that is beneficial to the sender of the signal, a process called sensory exploitation [START_REF] Ryan | Sexual selection, receiver biases, and the evolution of sex differences[END_REF][START_REF] Shaw | Phylogenetic tests of the sensory exploitation model of sexual selection[END_REF]. In almost all known cases, mating signals that arise through sensory exploitation capitalize on attractive stimuli, like food cues [START_REF] Fleishman | The influence of the sensory system and the environment on motion patterns in the visual displays of anoline lizards and other vertebrates[END_REF][START_REF] Rodd | A possible nonsexual origin of mate preference: are male guppies mimicking fruit?[END_REF][START_REF] Proctor | Sensory exploitation and the evolution of male mating behaviour: a cladistic test[END_REF][START_REF] Gilg | Isolation and functional expression of an animal geranyl diphosphate synthase and its role in bark beetle pheromone biosynthesis[END_REF] or refuges from predators [START_REF] Christy | Attractiveness of sand hoods built by courting male fiddler crabs, Uca musica: test of a sensory trap hypothesis[END_REF], which increases the likelihood of gaining the receiver's attention and motivating the receiver to approach. In the context of mate attraction, signals that tap into pre-existing sensory specializations for predator defense are, understandably, extremely rare (but see [START_REF] Nakano | Evolution of deceptive and true courtship songs in moths[END_REF]). An important step in determining whether a signal arose through sensory exploitation is demonstrating that the response to the signal preceded the appearance of the signal itself, a step that requires a phylogenetic framework, which is not always available [START_REF] Shaw | Phylogenetic tests of the sensory exploitation model of sexual selection[END_REF]. Furthermore, we seldom understand the neural origins of sensory biases, despite the important role that neural tuning and sensitivity plays in this process (but see [START_REF] Gerhardt | Pre-existing sensory biases in the spectral domain in frogs: empirical results and methodological considerations[END_REF]).

In this study, we combine behavioral and neural data in a phylogenetic context to assess the role of sensory exploitation in the evolution of an unusual communication signal in crickets.

Female field crickets (Gryllidae: Gryllinae) approach low-frequency sounds typical of male calling song (5 kHz) and avoid high-frequency sounds (>10 kHz) like the echolocation calls of predatory bats [START_REF] Wyttenbach | Categorical perception of sound frequency by crickets[END_REF][START_REF] Weber | Phonotactic behavior of walking crickets[END_REF]. Two ascending auditory interneurons, AN1 and AN2, forward activity from auditory afferents in the thorax to the brain [START_REF] Schildberger | Central auditory pathway: neuronal correlates of phonotactic behavior[END_REF]. AN1 is narrowly tuned to the low frequencies of the calling song, and it initiates positive phonotaxis [START_REF] Schildberger | The function of auditory neurons in cricket phonotaxis. I. Influence of hyperpolarization of identified neurons on sound localization[END_REF]. AN2 is tuned to higher sound frequencies and triggers avoidance behavior in response to bat calls [START_REF] Marsat | Bursting neurons and ultrasound avoidance in crickets[END_REF]. Eneopterine males (Gryllidae: Eneopterinae) are the only crickets known to generate calling songs with intense high-frequency harmonics [START_REF] Robillard | A shift toward harmonics for high-frequency calling shown with phylogenetic study of frequency spectra in Eneopterine crickets (Orthoptera, Grylloidea, Eneopteridae)[END_REF]. In one derived tribe, the Lebinthini, one of the higher-frequency harmonics has become the dominant frequency of the call, and this harmonic is ultrasonic (>20 kHz) in some species [START_REF] Robillard | A shift toward harmonics for high-frequency calling shown with phylogenetic study of frequency spectra in Eneopterine crickets (Orthoptera, Grylloidea, Eneopteridae)[END_REF]. Considering that in closely related field cricket species [START_REF] Chintauan-Marquier | Laying the foundations of evolutionary and systematic studies in crickets (Insecta, Orthoptera): a multilocus phylogenetic analysis[END_REF], high-frequency sounds provoke avoidance behavior [START_REF] Wyttenbach | Categorical perception of sound frequency by crickets[END_REF][START_REF] Thorson | Auditory behavior of the cricket. II. Simplicity of calling-song recognition in Gryllus, and anomalous phonotaxis at abnormal carrier frequencies[END_REF], the use of high-frequency calling songs for mate pairing is surprising.

We considered two hypotheses for the origin of high-frequency calling songs in lebinthine crickets. Most of the energy in male field crickets calls is restricted to a low frequency band, but there is still significant energy in higher frequency harmonics [START_REF] Hung | Is significant acoustic energy found in the audible and ultrasonic harmonics in cricket calling songs?[END_REF]. Although these harmonics are not required for phonotaxis, female crickets of at least one species prefer and can more accurately locate song models when they contain these higher frequency harmonics [START_REF] Latimer | Song harmonic content as a parameter determining acoustic orientation behaviour in the cricket Teleogryllus oceanicus (Le Guillou)[END_REF]. Eneopterine crickets often live on plants instead of on the ground, and increasing the energy in the higher-frequency harmonics of calls might have provided a greater benefit to crickets locating mates in the threedimensional habitat of plants, compared to the less complex two-dimensional habitat of crickets that live on the ground. The high-frequency calls of lebinthine crickets could have been a result of selection for a call feature more suitable for their complex environment. This hypothesis predicts that female lebinthine crickets will preferentially select males with higher frequency calls. Highfrequency calls could have also arisen by sensory exploitation if males were exploiting an antipredator behavior in females. This second hypothesis predicts that female lebinthine crickets demonstrate anti-predator-like behavior in response to male calls.

Female cricket behavior in response to male calling song

To test these hypotheses, we first analyzed and compared female behavior during male calling songs in five cricket species within the family Gryllidae (Figure 1). Two species have lowfrequency calling songs: Gryllus bimaculatus, an outgroup species from the subfamily Gryllinae, and Nisitrus vittatus, from the tribe Nisitrini within the Eneopterinae. Three species have highfrequency songs: Cardiodactylus muria, Agnotecous obscurus, and Lebinthus luae, all from the tribe Lebinthini within the Eneopterinae. Positive phonotaxis to the male calling song is well documented in field crickets (Gryllinae) [START_REF] Wyttenbach | Categorical perception of sound frequency by crickets[END_REF][START_REF] Weber | Phonotactic behavior of walking crickets[END_REF]. In arena experiments, we found that female N. vittatus also demonstrated a positive phonotactic response by walking to a speaker broadcasting the male calling song (Figure 2). Females of the three lebinthine species, however, did not walk at all when presented with the species-specific calling song (Figure 2). Instead, they remained stationary during male calling song but produced a vibrational response by jerking their body after each male call. We also observed female lebinthine crickets producing vibrational responses to male calls in the wild while remaining perched on leaves (Movie S1). We never observed vibrational responses in N. vittatus during playbacks of male calling song (Figure 2).

To determine how male and female lebinthine crickets find each other, we made observations of couples (one male and one female) for two species (C. muria and A. obscurus) in a cylindrical mesh arena. Observation sessions were conducted under red light and lasted for 20 minutes from the time that the male started calling. For C. muria, 10 of 13 couples tested located each other, and for A. obscurus, 3 of 5 couples located each other. In all cases, the behavior of males and females were consistent. The male lebinthine crickets produced calls and then walked a short distance in the arena before producing another call. Females did not walk, but remained stationary and produced vibrational signals. For communicating couples, it was always the calling male that walked to the stationary female. This is different from other species of crickets in which males remain stationary while calling and are approached by females [START_REF] Latimer | Song harmonic content as a parameter determining acoustic orientation behaviour in the cricket Teleogryllus oceanicus (Le Guillou)[END_REF][START_REF] Brown | Mate choice in tree crickets and their kin[END_REF][START_REF] Loher | Reproductive behavior[END_REF][START_REF] Shaw | Acoustic preference functions and song variability in the Hawaiian cricket Laupala cerasina[END_REF][START_REF] Forrest | Phonotaxis and calling in Puerto Rican mole crickets (Orthoptera: Gryllotalpidae)[END_REF]. We also observed male lebinthine crickets in the wild alternating between singing and walking and eventually tracking down a nearby stationary female producing vibrational signals in response to his calls (Movies S2, S3).

We then assessed the frequency tuning and timing of the female response relative to the male call. When song models that contained only one of the harmonics of the male call (6, 12, or 18 kHz) were broadcast, females of the low-frequency eneopterine species N. vittatus only showed phonotaxis to the 6 kHz song model, but not to the 12 or 18 kHz song models (arena tests; N=2).

The vibrational signals produced by female lebinthine crickets in response to computer generated male calls were broadly tuned to high frequencies (10-20 kHz; Figure 2) and the tuning was similar to that of the AN2 neuron and negative phonotaxis in G. bimaculatus and other field cricket species [START_REF] Schildberger | Temporal selectivity of identified auditory neurons in the cricket brain[END_REF][START_REF] Boyd | Two-tone suppression and song coding by ascending neurones in the cricket Gryllus campestris L[END_REF][START_REF] Jeffery | Selective processing of calling songs by auditory interneurons in the female cricket, Gryllus pennsylvanicus: possible roles in behavior[END_REF]. The females responded within a narrow range of latencies after the end of each male call, and the latency differed between species (Table 1).

The acoustic startle reflex in crickets

We hypothesized that the origin of the female vibrational response could be a startle reflex, a rapid jerking and freezing movement in response to a sudden stimulus, that is ubiquitous in many animals [START_REF] Koch | The neurobiology of startle[END_REF] including insects [START_REF] Werner | Responses of non-flying moths to ultrasound: the threat of gleaning bats[END_REF][START_REF] Friedel | The vibrational startle response of the desert locust Schistocerca gregaria[END_REF]. To test if the acoustic startle response preceded the appearance of the vibrational reply, a condition required for sensory exploitation, we measured the behavioral responses of four species of field crickets (Gryllinae) and two species of eneopterines to a high-amplitude (90 dB SPL) sound pulse at two frequencies: 6 kHz (similar to the dominant frequency in the calls of most field crickets) and 14 kHz (similar to the dominant frequency in the calls of many lebinthine species) (Table 1). All four field cricket species reliably generated short-latency substrate vibrations in response to the 14 kHz stimulus, indicating an acoustic startle reflex (ASR), but never in response to the 6 kHz stimulus. The latencies between the onset of sound and the onset of the ASR in these four species are similar to the latencies recorded for the bat-avoidance response by flying crickets (35-66 ms) [START_REF] Nolen | Phonotaxis in flying crickets. I. Attraction to the calling song and avoidance of bat-like ultrasound are discrete behaviors[END_REF], a behavior known to be triggered by AN2 [START_REF] Marsat | Bursting neurons and ultrasound avoidance in crickets[END_REF]. In contrast, the lebinthine species C. muria did not show any vibrational response (ASR) to these sound stimuli (Table 1); instead, tests using different durations and repetition rates of highfrequency sound pulses indicate that they require the species-specific male calling pattern to produce a vibrational reply (Figure S1). The latencies for the vibrational reply in lebinthine species are also considerably longer than what is typical for acoustic startle (Table 1).

The neural basis for mate pairing behavior in lebinthine crickets

If the female behavior evolved from a startle response, we would expect the lebinthine AN2 to have the same frequency tuning as the female vibrational reply. We recorded the spike activity of ascending auditory neurons (ANs) in the lebinthine C. muria and in the field cricket G. bimaculatus (Figure 3). Extracellular recordings from the connectives between the prothoracic and the subesophageal ganglia in G. bimaculatus showed similar activity levels in response to 5 and 14 kHz sound pulses due to AN1 and AN2 activity, respectively (Figure 3A). Connective recordings in the lebinthine species, however, showed no neural activity in response to 5 kHz pulses and a high level of activity in response to 14 kHz pulses (Figure 3A). Therefore, there is no lowfrequency sound response forwarded to the brain in C. muria that corresponds with the narrow AN1 tuning known from field crickets [START_REF] Schildberger | Central auditory pathway: neuronal correlates of phonotactic behavior[END_REF]. Intracellular recordings and staining experiments were conducted to reveal the tuning of individual auditory interneurons in the brains of these two cricket species (Figure 3B, Figure S2, Figure S3). In G. bimaculatus, we could unambiguously discriminate between AN1 (N=20) and AN2 (N=20) based on their characteristic frequency tuning and morphology. All ascending (N=11) and local (N=6) auditory interneurons recorded in 11 C. muria, however, were only sensitive to high-frequency sounds with a threshold curve similar to AN2 of G. bimaculatus. The threshold curves of ANs in C. muria were consistent between animals and closely matched the thresholdtuning curve for the female vibrational response (Figure 3B). Thus, both the extracellular and intracellular recordings demonstrate that the ascending auditory neurons in C. muria only respond to high-frequency sounds. Successful intracellular stainings of ANs in the brains of four C. muria did not show a clear AN1-like or AN2-like morphology as in Gryllus (Figure 3C). Therefore we speculate that either the AN1 has been lost in the lebinthine species or its frequency tuning has shifted to that of the AN2.

DISCUSSION

Our results support the hypothesis that sensory exploitation in the communication system of lebinthine crickets led to the transformation of a startle response to a mate-pairing signal (Figure S4). This hypothesis suggests that, starting with mate pairing typical for field crickets in which females approach singing males, the high-frequency components of the calling songs in eneopterine crickets increased over evolutionary time until the male song triggered an ASR in females of the ancestral lebinthine crickets. Male lebinthine crickets that followed substrate vibrations generated by the female had the advantage of finding mates without waiting to be selected by one and possibly reducing their risk from acoustically orienting predators and parasitoids [START_REF] Sakaluk | Gecko phonotaxis to cricket calling song: a case of satellite predation[END_REF][START_REF] Müller | Death comes suddenly to the unprepared: singing crickets, call fragmentation, and parasitoid flies[END_REF]. Likewise, females potentially reduced their risk of predation by remaining stationary. Potential costs to lebinthine females producing ASRs, such as attracting vibration-sensing predators [START_REF] Barth | Spiders of the genus Cupiennius Simon 1891 (Araneae, Ctenidae). II. On the vibratory environment of a wandering spider[END_REF], might have selected for females that responded to a narrower range of highfrequency stimuli (i.e. only the calls of conspecific males) as opposed to any high-frequency sound.

As the vibrational response was co-opted into a true communication signal, individuals of lebinthine species lost the typical cricket ASR to any high-frequency sound pulse. Thus, what started as sensory exploitation by males of a startle reflex (ASR) in females evolved into a new auditory-vibratory communication system with females selectively producing vibrational signals in response to the calls of conspecific males.

The behavior seen in lebinthine females and the presence of phonotaxis in ancestral taxa suggests that female phonotaxis has been lost in the Lebinthini. Female phonotaxis to male calls has been documented in three of seven cricket families, including one of the most basal groups (the Gryllotalpidae) and three subfamilies within the family Gryllidae (Fig. 1A). Because of the lack of knowledge about the character state in some families, it is difficult to assess if phonotaxis is the ancestral condition for all crickets. However, the most parsimonious explanation for the taxa included in this study (Gryllinae and Eneopterinae) is that the ancestral condition of female phonotaxis was lost once in the ancestor of the Lebinthini, as opposed to female phonotaxis evolving twice independently in the Gryllinae and Nisitrini.

The behavioral pattern seen in female lebinthine crickets is typical of insects that communicate with acoustic duets in which the male call is followed by a female reply in a species-specific time window [START_REF] Bailey | Insect duets: underlying mechanisms and their evolution[END_REF]. In lebinthines, however, males and females use different modalities and communication channels (acoustic and vibrational signals, respectively). Vibration-based communication through plant substrates is widespread across many insect groups, including the Orthoptera [START_REF] Cocroft | The behavioral ecology of insect vibrational communication[END_REF]. The dominant frequencies of the vibrational reply produced by lebinthine crickets (C. muria: 38 ± 3 Hz; A. obscurus: 48 ± 5 Hz; L. luae: 84 ± 4 Hz; mean ± SEM) are in the range of those produced by non-acoustic crickets for vibrational communication [START_REF] Stritih | The role of frequency in vibrational communication of Orthoptera[END_REF] and activate the vibration-sensitive subgenual organ in the legs of crickets [START_REF] Dambach | The sense of vibration in crickets. I. Threshold measurements from the leg nerves of freely-moving animals[END_REF]. Furthermore, plants transmit vibrations very well compared to the ground substrate of field crickets [START_REF] Cocroft | The behavioral ecology of insect vibrational communication[END_REF]. Therefore, these vibrational signals are well suited for detection by eneopterine crickets.

Females of some field cricket species can more accurately locate male song that includes highfrequency harmonics [START_REF] Latimer | Song harmonic content as a parameter determining acoustic orientation behaviour in the cricket Teleogryllus oceanicus (Le Guillou)[END_REF], and this could have selected for greater energy in higher frequency harmonics in eneopterines due to their tendency to live on the more complex environment of plants rather than the ground. Because vibratory cues can accompany male singing [START_REF] Weideman | Influence of vibratory signals on the phonotaxis of the gryllid Gryllus bimaculatus DeGeer (Ensifera: Gryllidae)[END_REF] and courtship behavior [START_REF] Bell | Multimodal communication by the black-horned tree cricket, Oecanthus nigricornis (Walker) (Orthoptera: Gryllidae)[END_REF] in crickets, and male field crickets are known to phonotactically approach other singing males [START_REF] Mccarthy | Phonotactic behavior of male field crickets (Gryllus texensis) in response to acoustic calls from conspecific males[END_REF], territoriality or competition might have been the original impetus for males to approach the vibrations produced by startled females.

The function of the high-frequency ASR in perched insects is unknown because it provides no protection against attacking bats. It is possible that it is a by-product of the bat avoidance response during flight and has no adaptive function or significant cost in this context. Both the ASR in crickets and vibrational signal in female lebinthines are most likely mediated by activity in the AN2 interneuron, which triggers cricket avoidance steering in flight. The latencies of the vibrational reply in female lebinthines, however, are significantly longer than what is observed for ASRs (Table 1), and the vibrational replies are tuned to the temporal pattern of the species-specific male call, as is seen in other duetting insects [START_REF] Bailey | Insect duets: underlying mechanisms and their evolution[END_REF]. As AN1 is crucial for calling song pattern recognition in field crickets [START_REF] Schöneich | An auditory feature detection circuit for sound pattern recognition[END_REF][START_REF] Kostarakos | Calling song recognition in female crickets: temporal tuning of identified brain neurons matches behavior[END_REF], its tuning in lebinthine crickets might have shifted to higher frequencies to support pattern recognition. Further experiments are needed on the tuning, morphology, and targets of the auditory ANs and local brain neurons across eneopterine cricket species with established phylogenetic relationships to assess in more detail how the function of these neurons changed over evolutionary time in the eneopterine clade.

The origins of communication signals have long fascinated evolutionary biologists, and

multiple potential mechanisms for these origins have been proposed [START_REF] Bradbury | Principles of Animal Communication[END_REF][START_REF] Darwin | The expression of the emotions in man and animals[END_REF]. Evidence from many well-documented cases over the past 25 years suggest that sensory biases in receivers is a relatively common origin for novel communication signals [START_REF] Fleishman | The influence of the sensory system and the environment on motion patterns in the visual displays of anoline lizards and other vertebrates[END_REF][START_REF] Rodd | A possible nonsexual origin of mate preference: are male guppies mimicking fruit?[END_REF][START_REF] Proctor | Sensory exploitation and the evolution of male mating behaviour: a cladistic test[END_REF][START_REF] Gilg | Isolation and functional expression of an animal geranyl diphosphate synthase and its role in bark beetle pheromone biosynthesis[END_REF][START_REF] Christy | Attractiveness of sand hoods built by courting male fiddler crabs, Uca musica: test of a sensory trap hypothesis[END_REF]. In almost all cases of exploitation of sensory biases for mate finding, senders converge on properties of environmental cues that are attractive to receivers due to the clear benefits this has for the sender. A particularly interesting aspect of the lebinthine communication system is that, unlike other examples in which senders increase the probability that receivers will move towards them, male lebinthine crickets induced a response in females (the ASR) that then evolved into a new communication signal itself (the vibrational reply). Our phylogenetic, behavioral, and neurophysiological evidence demonstrates that sensory exploitation of an aversive cue can also evolve into a classic communication system in which both senders and receivers benefit, and that this unexpected origin might be more common than previously appreciated.

SUPPLEMENTAL INFORMATION

Supplemental Information includes Supplemental Data (4 figures and 3 movies) and Supplemental Experimental Procedures.

AUTHOR CONTRIBUTIONS

HtH conducted behavioral and extracellular neural recording experiments. SS conducted intracellular recording and staining experiments. TR observed and recorded videos of behavior in the field. All authors contributed to the conception, design and writing of this study.

FIGURES Figure 1. Phylogenetic relationships, male calling song features, and female behavior in five cricket species.

(A) Inferred phylogenetic tree of cricket families and subfamilies with a simplified topology, based on a large-scale analysis (205 species) using seven nuclear and mitochondrial molecular markers [START_REF] Chintauan-Marquier | Laying the foundations of evolutionary and systematic studies in crickets (Insecta, Orthoptera): a multilocus phylogenetic analysis[END_REF] showing Bayesian posterior probabilities corresponding to each node. Families and subfamilies in which female phonotaxis to male calls has been documented are given in green [20-24; this study], families and subfamilies in which it is unknown if females perform phonotaxis to male calls are given in grey with dashed lines, families in which acoustic communication has been lost are given in orange [START_REF] Henderson | Biology of the myrmecophilous cricket, Myrmecophila manni (Orthoptera: Gryllidae)[END_REF], and the subfamilies which have acoustic

bimaculatus) and a lebinthine species (C. muria).

(A) Standardized relative neural activity in response to sound measured by extracellular recordings at the neck connective (see Supplemental Experimental Procedures for an explanation of the measurement of neural activity, a.u. = arbitrary units; mean + SD, N = 6 for each species, ***: p<0.001, student t-test).

(B) Threshold tuning curves for ascending neurons measured using intracellular recording methods (mean -SD; N=20 for Gryllus AN1 and AN2, N=11 for Cardiodactylus ANs). For comparison of thresholds, the black line represents thresholds for female vibrational responses to Cardiodactylus male call models. See Figure S2 for example recordings and Figure S3 for individual tuning curves.

(C) Axonal branches of ascending auditory neurons in the brain, reconstructed from intracellular stainings.

Table 1. Behavioral responses to sound stimuli of females in eight cricket species.

Latency values are means ± SEM and different letters (superscript) indicate significantly different groups (Kruskal-Wallis test, Bonferroni correction for post hoc tests; G. rubens and A. domesticus were excluded from statistical analysis due to small sample sizes). See Figure S4 for a hypothesized sequence of events leading to the evolution of vibrational signaling in lebinthine crickets from an ASR precursor. The average values for pulse duration, pulse period (time from the start of one pulse to the start of the next), and call duration (time from the start of the first pulse to the end of the last pulse) were used as a guide to generate calling song models at single frequencies. The C. muria call model consisted of a single 50 ms pulse repeated every 5 s (Fig. 1). The L. luae call model had two parts separated by 380 ms of silence: 1) a series of fifteen 20 ms pulses with a pulse period of 200 ms, and 2) a series of twenty-five 15 ms pulses with a pulse period of 25 ms. Total call duration was thus 3.815 s and calls were repeated every 15 s. The A. obscurus call model consisted of a single chirp containing fifty 16 ms pulses with a pulse period of 30 ms. Total call duration was thus 1.486 s and calls were repeated every 6.5 s.

Subfamily

Observations of female behavior in response to male calling song: We observed cricket behavior under red light in a cylindrical fiberglass mesh arena (47 x 33 cm, L x D) placed in an anechoic chamber lined with sound attenuating foam. For phonotaxis trials, two speakers were placed in the anechoic chamber, one on each end of the cylindrical cage. Virgin female crickets were placed individually in the cage (N. vittatus, N=4; C. muria, N=8; A. obscurus, N=7; L. luae, N=9). A recording of species-specific male calling song was broadcast at 65 and 75 dB peSPL at the center of the arena for 20 minutes each from different ends of the cage, during which time the behavior of the cricket was observed. Behavior was scored as phonotaxis if the female walked within 5 cm of the end of the cage during playback of male calling song. Interactions between male and female lebinthine crickets were observed under red light in the same cylindrical arena described above (C. muria, N=13 pairs; A obscurus, N=5 pairs). We waited for males to start singing or we stimulated male calling by broadcasting species-specific male song, and observed the couple for at least 20 minutes, or until the male and female found each other in the cage. During this time, we made detailed observations of the behavior of the male (calls, direction of walking behavior) and female (vibrational replies, walking behavior). and 3) staggered frequencies [START_REF] Shaw | Phylogenetic tests of the sensory exploitation model of sexual selection[END_REF][START_REF] Robillard | A shift toward harmonics for high-frequency calling shown with phylogenetic study of frequency spectra in Eneopterine crickets (Orthoptera, Grylloidea, Eneopteridae)[END_REF][START_REF] Gerhardt | Pre-existing sensory biases in the spectral domain in frogs: empirical results and methodological considerations[END_REF][START_REF] Latimer | Song harmonic content as a parameter determining acoustic orientation behaviour in the cricket Teleogryllus oceanicus (Le Guillou)[END_REF][START_REF] Rodd | A possible nonsexual origin of mate preference: are male guppies mimicking fruit?[END_REF][START_REF] Marsat | Bursting neurons and ultrasound avoidance in crickets[END_REF][START_REF] Schildberger | The function of auditory neurons in cricket phonotaxis. I. Influence of hyperpolarization of identified neurons on sound localization[END_REF]12 and 18 kHz). The order for these three trials was randomized for each cricket.

Measuring vibrational response tuning

The threshold for the female vibrational response to male calls was measured as the lowest sound level at which vibrations were registered by the accelerometer. The threshold reported for each animal was the minimum threshold of these three trials. Response latency was measured as the time from the start of the last pulse in the male call to the start of the female vibrational signal. The frequency with the most energy in the vibrational reply was measured from power spectra generated in SASLabPro software (Avisoft Bioacoustics, Berlin, Germany).

Measuring acoustic startle responses: Female crickets were placed individually in a cylindrical metal mesh cage (17 x 9 cm, L x D).

The cage was placed between a speaker and a 1/2" microphone and sound pulse amplitude was calibrated at the center of the cage. Puretone sound pulses of 100 ms duration (with 5 ms rising and falling ramps) and 90 dB SPL at the position of the cricket were presented at 6 or 14 kHz sound frequency. Sound stimuli were broadcast in random order for each cricket with 5 minutes of silence between stimuli. The beam of a Laser Doppler Vibrometer (PSV-400; Polytec, Waldbronn, Germany) was focused onto a piece of reflective foil on the cage, allowing for the detection of low amplitude vibrations in the cage generated by small movements of the cricket in response to sound. The presence or absence of vibrations within 500 ms following the stimulus was recorded, and the latency of the response was measured as the time from the start of the sound stimulus to the start of the vibration signal.

  communication but lack phonotaxis are given in magenta [this study]. (B) Cricket species investigated in this study and photographs of males. (C) Oscillograms of complete male calling song (upper panel) and oscillograms of individual sound pulses (middle panel) with power spectra (left lower panel) and spectrograms (right lower panel) for each species. Stars highlight the dominant frequency of the call. (D) Category of female behavioral responses (phonotaxis or vibrational response) to conspecific male calling song. See Movies S1, S2, and S3 for examples of the lebinthine male calling and searching behavior and female vibrational replies.

Figure 2 .

 2 Figure 2. Female behavioral responses to male calls in four cricket species.

Figure 3 .

 3 Figure 3. Neural response of ascending interneurons to sound for a field cricket (G.

Figure S1, related to Figure 2 .

 2 Figure S1, related to Figure 2. Specificity of the Cardiodactylus muria female vibrational reply to call duration and call interval. All sound stimuli were 13 kHz sound pulses broadcast at 75 dB peak-equivalent SPL at the center of the cage. (A) Percentage of females producing a vibrational reply to sound pulses of varying durations repeated every 4 s (average male call duration: 44.8 ± 1.5 ms, N=6). (B) Percentage of sound pulses eliciting a female vibrational response for six 50 ms sound pulses repeated at different periods (average male calling song period: 5.6 ± 0.6 s, N=6). Black bars are median values; grey boxes are 10-90% percentiles ranges.

Figure S2, related to Figure 3 .

 3 Figure S2, related to Figure 3. Tuning curves and intracellular recordings of auditory ascending neurons in three cricket species. (A) Tuning curves showing the average number of action potentials produced in response to a 20 ms sound pulse at a range of frequencies and amplitudes (N = number of animals, n = number of sound presentations). (B) Examples of intracellular recordings of ascending neurons during playback of 5 kHz and 14 kHz sound pulses with increasing amplitude.

Figure S3, related to Figure 3 .

 3 Figure S3, related to Figure 3. Threshold tuning curves for auditory neurons based on intracellular recordings in the brains of three cricket species. (A) Ascending interneurons. (B) Brain neurons. For Gryllus and Cardiodactylus, each line is a recording from a different animal, for Lebinthus each line is a different recording from the same animal. Threshold was defined as an average response of at least one action potential. The decrease in threshold at ca. 4 kHz seen in L. luae is a feature that is also observed in recordings of AN2 neurons from several field cricket species (Gryllus campestris [S1], Gryllus pennsylvanicus [S2], Teleogryllus commodus [S3]).

Figure S4, related to Table 1 .

 1 Figure S4, related to Table 1. Hypothesized evolutionary sequence of events leading from an acoustic startle response to a vibrational signal in lebinthine crickets. Arrows indicate the direction of cricket movement, tildes indicates the production of substrate vibrations.

:

  Unmated females of three eneopterine cricket species (C. muria, A. obscurus, L. luae) were placed in a vertically oriented cylindrical metal mesh cage (17 x 9 cm, L x D). Females of these species always perched on the vertical wall of the cage. An accelerometer (type 4393V, Brüel and Kjaer, Naerum, Denmark) was attached to the side of the cage and the cage was placed in the same sound-attenuating chamber described above. Accelerometer signals were amplified (Nexus Conditioning Amplifier, Brüel & Kjaer, Naerum, Denmark) and recorded using a National Instruments AD board (PCI-MIO-16E4; NI, Austin, TX, USA) and Labview software. Song models for each species were generated at 2, 4, 6, 8, 10, 12, 14, 16, 18 and 20 kHz. For each frequency, the call model was broadcast at 21 amplitudes (40-80 dB peSPL increasing in 2 dB steps). Each female was tested three times over three days with different orders for the frequencies: 1) increasing frequencies (2-20 kHz), 2) decreasing frequencies (20-2 kHz),

Extracellular neural recordings :

 recordings Extracellular neural recordings were made with a double-hook electrode on the connective ipsilateral to the sound source and anterior to the prothoracic ganglion [S5]. The crickets were fixed ventral side up to a metal base and the front legs were waxed to wires holding them in a natural position [S6]. To reduce any neural activity unrelated to sound stimuli, both connectives posterior to the prothoracic ganglion and the contralateral connective anterior to the prothoracic ganglion were cut, and the ipsilateral connective was pinched with forceps anterior to the recording electrode. Sound stimuli were 50 ms pulses (with 5 ms rising and falling ramps) at 2, 3, 4, 5, 6, 8, 10, 12, 14, 16, 18, and 20 kHz with 200 ms silence between pulses, adjusted to 80 dB peSPL at the position of the cricket. The stimulation sequence was repeated 1000-2000 times for each cricket. Neural signals were amplified (DC amplifier BA-01X; NPI, Tamm, Germany) and digitally recorded with a National Instruments AD board (PCI-MIO-16E4; NI, Austin,

ACKNOWLEDGEMENTS

We are grateful to R. Calsbeek, M. McPeek, J. Ratcliffe, L. Symes, and A. Vacca for helpful comments on earlier versions of this manuscript. We also thank F. Legendre, H. Jordan, M.-K.

Tan and Y. Suhardjono for help with establishing the eneopterine cricket colonies. In Singapore, adult specimens were collected from the wild under National Parks Board permits (numbers NP/RP924 and NP/RP10-073a). Funding for this project was provided by a Natural Sciences and Engineering Research Council of Canada postdoctoral fellowship and a John Stanley Gardiner Fund Travel Grant to HtH, and a Biotechnology and Biological Sciences Research Council grant (UK) to BH. It also benefitted from grants by Actions Transversales MNHN ʻFormes Possibles, Formes Réalisées' (V. Bels, P.-H. Gouyon), 'Emergences' (S. Peigné, P. Janvier), and "Génomique et collection" (S. Samadi, R. Debruyne). Additional video data presented in this study were obtained in the framework of "Our Planet Reviewed Papua-New-Guinea 2012-2013" set up by Pro-Natura International, the Muséum national d'Histoire naturelle (MNHN, France), the Institut de Recherche pour le Développement (IRD, France) in partnership with the Royal Belgian Institute of Natural Sciences, the New Guinea Binatang Research Center, the University of Papua New Guinea, and the Divine Word University of Madang and with core funding of Prince Albert II of Monaco Foundation, the Stavros Niarchos Foundation, the Total Foundation, the Fondation d'entreprise EDF, the Fonds Pacifique, Spiecapag, Entrepose Contracting, the New-Caledonia Government, the Reef Foundation and the Belgian National Lottery.

TX, USA) for storage and off-line analysis with Neurolab software [S7]. To extract the responses to sound from background neural activity, the absolute value of the voltage signal was used and averaged for each stimulus. The background activity was set to zero and the area under the neural response was calculated as a measure of overall neural activity in response to a sound pulse [S6]. To standardize the data across individual crickets, neural activity is presented as a relative response (area for the frequency divided by the mean area across all frequencies) (Fig. 3). We used t-tests to assess differences in neural activity between 5 and 14 kHz because these are frequencies that stimulate activity almost exclusively in AN1 and AN2, respectively.


Stylianos Panagiotakis
email: s.panagiotakis@inn.demokritos.gr

Barbara Mavroidi
email: bmavroidi@bio.demokritos.gr

Alexandros Athanasopoulos

Antonio Ricardo Gonçalves

Loïc Bugnicourt-Moreira

Theo Regagnon
email: tregagnon@hotmail.fr

Nikos Boukos
email: n.boukos@inn.demokritos.gr

George Charalambidis

Athanasios G Coutsolelos

Mantas Grigalavicius

Theodossis A Theodossiou
email: theodossis.theodossiou@rr-research.no

Kristian Berg
email: kristian.berg@rr-research.no

Catherine Ladavière
email: catherine.ladaviere@univ-lyon1.fr

Maria Pelecanou

Konstantina Yannakopoulou
email: k.yannakopoulou@inn.demokritos.gr

Small anticancer drug release by light: Photochemical internalization of porphyrin-β-cyclodextrin nanoparticles

Keywords: cyclodextrin-porphyrin conjugates, drug delivery, photochemical internalization

Introduction

Carbohydrate-based drug delivery systems that upon light stimulation combine phototoxic and chemotoxic action in high synergy for tumor treatment are continuously sought [START_REF] Zhang | Carbohydrates based stimulus responsive nanocarriers for cancer-targeted chemotherapy: a review of current practices[END_REF]. Biocompatible, water soluble carrier systems can combine selectively and synergistically anticancer drugs with suitable photosensitizer (PS) molecules that upon illumination can induce formation of reactive oxygen species (ROS) that are deleterious to biological matter. The ideal combination would also be capable of targeting cell organelles as this is critical to achieve high complementary efficacy in inducing cancer cell death [START_REF] Kessel | Critical PDT theory II: Current concepts and indications[END_REF]. Photodynamic therapy (PDT), a modality relying on the preferential retention of PSs in solid tumor tissue, utilizes focused light, e.g. diode lasers, to initiate a photokilling process that spares healthy cells [START_REF] Dolmans | Photodynamic therapy for cancer[END_REF][START_REF] Agostinis | Photodynamic therapy of cancer: an update[END_REF]. Amongst the PSs used, porphyrins hold a prominent position, due to both favorable photophysical characteristics and relative molecular stability, compared to chlorins and bacteriochlorins [START_REF] Dabrowski | Engineering of relevant photodynamic processes through structural modifications of metallotetrapyrrolic photosensitizers[END_REF]. However, their very low aqueous solubility and concomitant loss of photoactivity due to aggregation is a major obstacle for bio-applications, but this is a manageable issue for photochemical internalization (PCI), an advanced intervention that relies on endocytosis and requires that the drug is entrapped exclusively into endocytic vesicles (endosomes, lysosomes) while the PS is anchored in their membranes. Irradiation using a sub-lethal PDT dose results in membrane rupturing and drug release leading to synergistic photo and chemo-toxicity of tumor cells [START_REF] Jerjes | Photochemical internalization for intracellular drug delivery. From basic echanisms to clinical research[END_REF]. PCI has been implemented using chiefly macromolecular toxins that are unable to penetrate the plasma membrane and are thus internalized by endocytosis, co-administered with a suitable photosensitizer [START_REF] Sultan | Disulfonated tetraphenyl chlorin (TPCS2a)-induced photochemical internalisation of bleomycin in patients with solid malignancies: a phase 1, dose-escalation, first-in-man trial[END_REF]. Consequently, examples using small molecule chemotherapeutics in PCI protocols are scarce.

Cyclodextrins (CDs) are cyclic carbohydrate oligomers that form inclusion complexes with hydrophobic drugs and solubilize them in water. Natural or specifically modified CDs are used extensively as excipients in drug formulations [START_REF] Saokham | Solubility of cyclodextrins and drug/cyclodextrin complexes[END_REF]. Simple porphyrin-CD covalent conjugates are attractive because they combine photoactivity with drug inclusion and ease of synthesis.

Although several conjugates have appeared in the literature as [START_REF] Mavridis | Porphyrinoid-cyclodextrin assemblies in biomedical research: an update[END_REF][START_REF] Singh | Glycosylated porphyrins, phthalocyanines, and other porphyrinoids for diagnostics and therapeutics[END_REF] including an example originating from this lab [START_REF] Theodossiou | Photochemical internalization of tamoxifens transported by a "Trojan-Horse" nanoconjugate into breast-cancer cell lines[END_REF][START_REF] Fraix | A multifunctional bichromophoric nanoaggregate for fluorescence imaging and simultaneous photogeneration of RNOS and ROS[END_REF], the importance of prior nanoparticle (NP) formation and optimization has neither been properly recognized nor investigated in detail, to our knowledge, We hypothesized that by connecting the highly photoactive hydrophobic 5,10,15,20tetraphenylporphyrin (TPP) via linker alkyl chains of variable length to hydrophilic βCD we would create stable, small NPs by self-assembly in water. The hydrophobic linkers are expected to further modulate the amphiphilicity of the neutral conjugates, promote their molecular plasticity and capacity for self-assembly and augment their ability for encapsulation and transport of small anticancer drugs. If suitably sized, the photosensitive NPs will target endosomes/lysosomes of cells by endocytosis, carrying the bound drugs along. Apart from tamoxifen, an anticancer drug with multiple phenyl groups that match the βCD cavity size [START_REF] Aggelidou | Protoporphyrin IX-β-cyclodextrin bimodal conjugate: nanosized drug transporter and potent phototoxin[END_REF], gemcitabine, although unsuitably structured, could also be effective if first converted into its N-adamantyl pro-drug. Both were expected to bind tightly enough to NPs to ensure co-delivery to lysosomes. The proposed approach would provide a widened choice of vehicles and small chemotherapeutics for the PCI technology.

Herein we use 2, 5 and 11-carbon chain linkers to connect TPP to βCD (Scheme 1) and demonstrate the successful formation of photoactive NPs that bind and transport the above mentioned small chemotherapeutics, specifically targeting the lysosomes of tumor cells. The NPs, devoid of dark toxicity, exert highly synergistic phototoxic and chemotoxic action upon illumination. Moreover, we employ photoactive liposomes as model vesicles in order to mimic organelles and understand the subcellular fate of our systems.

Results and Discussion

Synthesis and structural characterization

Photophysical characterization

UV-Vis spectra in DMF and in aqueous solution:

The spectra of the conjugates and the corresponding parent porphyrins in the monomerizing [START_REF] Shi | Porphyrin-based compounds and their applications in materials and medicine[END_REF] solvent DMF (Figs.

1A and 1B, respectively, Table B1) display characteristic absorption features [START_REF] Panagiotakis | Unsymmetrical, monocarboxyalkyl meso-arylporphyrins in the photokilling of breast cancer cells using permethyl-βcyclodextrin as sequestrant and cell uptake modulator[END_REF][START_REF] Mandal | Photophysical properties and electronic structure of porphyrins bearing zero to four meso-phenyl substituents: new insights into seemingly well understood tetrapyrroles[END_REF][START_REF] Ormond | Effects of substituents on the photophysical properties of symmetrical porphyrins[END_REF] with Soret (418.5 nm) and Q bands (500 -675 nm). The Soret band intensities of TPPOCNHbmC2 and TPPOCNHbmC5 drop by ~75% and ~25%, respectively, relative to that of TPPOCNHbmC11 (Fig. 1A) thus, the shorter the separation of the carbonyl group from the porphyrin π-system the higher the hypochromic effect observed. In phosphate buffer saline containing 4% DMSO (v/v), pH 7.4 (PBS) the spectra of the conjugates are not as severely affected, compared to their parent porphyrins while TPPOCNHbmC11 displays remarkable recovery of its absorbance (Figs. 1C vs 1D). The Soret bands are relatively symmetrical with less pronounced "shoulders" on both sides of λmax and narrower at FWHM by ~ 4 -38 nm, suggesting presence of rather uniform populations of species in PBS, compared to the parent porphyrins. (. _ . _ .) PBS with excess pMβCD (8 μM); H) TPPOCOOHCx, PBS (773 nm band arises from scattering of excitation light). The concentrations in DMF were adjusted to A = 0.02 at λexc = 515 nm to enable comparison with the spectra in PBS.

Emission spectra in DMF and in aqueous solution:

In DMF and λexc= 515 or 510 nm two characteristic emission bands at 653 and 717 nm are observed (Fig. 1E1,F1,G1, Fig. B15, Table B1). In PBS and λexc= 515 nm the intensities of these emission bands are significantly diminished (Fig. 1E2,F2,G2, Fig. B15C, Table B2), but still, two-, three-or ten-fold increased, compared to the parent TPPOCOOHCx (Fig. 1H). To assess the emission properties of the conjugates in monomeric form in aqueous solution, we formed strong inclusion complexes in situ with 2,3,6-per-O-methyl-βCD (pMβCD), that specifically binds the TPP moiety (Table B3) as previously demonstrated with the parent porphyrins [START_REF] Panagiotakis | Unsymmetrical, monocarboxyalkyl meso-arylporphyrins in the photokilling of breast cancer cells using permethyl-βcyclodextrin as sequestrant and cell uptake modulator[END_REF]. Up to 3-fold enhancement of the emission was registered (Fig. 1E3,F3,G3) owing to de-aggregation resulting in emission intensities reaching (TPPOCNHbmC11) or surpassing (TPPOCNHbmC2, TPPOCNHbmC5) those in DMF.

Fluorescence lifetime measurements in DMF and in aqueous solution:

Lifetimes (τ) were obtained from the best fit of the fluorescence decay data with either mono-or bi-exponential functions (Table 1, Fig. B16). In DMF the single species lifetime, τ, obtained is 5 to 20% reduced, compared to that of their parent porphyrins (τ ~ 11 ns). In PBS, two lifetime values, τ1 ~1.0 ns and τ2 ~4.6 ns, were calculated. Each τ1 and τ2 species contributes nearly 50% to the total population (entries 1-3). In contrast, in the respective porphyrins (entries 4-6) the % fraction of the τ1 population increases drastically with linker chain-length whereas the % of the τ2 populations decreases. These results demonstrate that the conjugates behave similarly, nearly irrespective of the linker chain-length, and are organized into two approximately equal populations of aggregated species, with longer τ2 values and increased % fraction, compared to the parent porphyrins.

Self-assembly into nanoparticles (NPs)

The above measurements strongly indicate the presence of mostly nanoparticles (NPs) rather than random aggregates in the aqueous solution. To optimize NP formation, solutions of the conjugates were prepared according to "good-bad" methodology [START_REF] Karikis | Self-assembly of (borondipyrromethane)-diphenylalanine conjugates forming chiral supramolecular materials[END_REF]: TPPOCNHbmCx were dissolved in the "good-chaotropic" solvent DMSO and then diluted in the "bad" solvent PBS, reaching a final solution of PBS with 4% DMSO (v/v), pH 7.4 and were allowed to self-assemble at room temperature (r.t.) for 24 h.

Scanning and Transmission electron microscopies (SEM, TEM).

The SEM image of the NP assemblies formed by TPPOCNHbmC11, initially examined at 100 μM concentration, revealed three predominant populations of round, granular NPs (Fig. B17A1) with average diameters ~500 nm, ~200-300 nm and ~50-100 nm. However, at 20 μM and as TEM images (Fig. 2A and Figs. B17A2,A3) confirm, smaller, rather uniform nanospheres with average diameters of ~70 nm were formed. Few larger particles (~200 -350 nm, Fig. B17A2) were observed. Imaging did not require any contrasting agent, suggesting that the NPs may be relatively compact and dense. The EDX experiments (Fig. B17B) confirm the identity of the NPs. The corresponding TEM images of TPPOCNHbmC5 revealed a rather diffuse material on the grid (data not shown) suggesting that these NPs are more stable in the aqueous solution. 2), irrespective of the linker between TPP and βCD, in contrast to their parent porphyrins (entries 4-6, Table 2). Note that the DLS raw data (% intensity vs size) are inevitably affected by the absorption of the porphyrin moieties at the detection wavelength (633 nm)

and by the few large NPs that scatter intensely. The conjugates, therefore, seem to share a common self-organization mode. Evidently, the considerable amphiphilic character of the porphyrin-βCD dyad provides π-π stacking domains via the strongly hydrophobic TPP moieties, while the hydrophilic βCD cups are exposed. The NPs of TPPOCNHbmC2 and TPPOCNHbmC5 are stable for 72 h in PBS whereas the NPs of TPPOCNHbmC11 are stable for 48 h and then increase in size (Fig. B17C3). Among other properties, size affects the entrance, traffic and localization of NPs in cells. NPs of ~50 nm are most suitable for endocytic cell uptake, while NPs of greater dimensions are either less internalized or reside on cell membranes, the mechanisms additionally depending on the surface properties of the NPs [START_REF] Dolai | Nanoparticle size effects in biomedical applications[END_REF]. Thus, the present NPs seem suitably sized and sufficiently stable for cell experiments.

 iii Polydispersity Index; iv Tamoxifen (2-[4-[(Z)-1,2-diphenylbut-1-enyl]phenoxy]-N,N-dimethylethanamine) citrate; v
Gemcitabine hydrochloride; vi 1-Adamantyl-anecarboxylic acid.

Theoretical calculations for self-assembly.

The geometries of the conjugates were optimized using crystallographic coordinates for βCD and porphyrin moieties [START_REF] Giastas | Molecular structures of the inclusion complexes β-cyclodextrin/1,2-bis(4-aminophenyl)ethane and β-cyclodextrin/4,4΄-diaminobiphenyl. Packing of dimeric β-cyclodextrin inclusion complexes[END_REF][START_REF] Charalambidis | A switchable self-assembling and disassembling chiral system based on a porphyrin-substituted phenylalanine-phenylalanine motif[END_REF] at the Molecular Mechanics level of theory using Avogadro software. The calculations showed that due to twisting of the aliphatic linkers near the βCD narrow opening, the maximum molecular dimensions of the optimal geometries of TPPOCNHbmC5 and TPPOCNHbmC11 become very similar, ~2.9 nm (Fig. B18), in support of the experimental results.

Anticancer drug inclusion in the NPs

Two, differently structured drugs were considered for loading in the NPs. Tamoxifen (2-[4-[(Z)-1,2diphenylbut-1-enyl]phenoxy]-N,N-dimethylethanamine) is a selective estrogen receptor modulator drug (SERM) prescribed as citrate salt (TamCit) to prevent and treat estrogen receptor positive breast cancer (Osborne, 1998). We have previously determined a substantial 1:1 binding constant (7.0 ±1.5x10 3 M -1 ) between βCD and N-desmethyltamoxifen (NDTAM) [START_REF] Aggelidou | Protoporphyrin IX-β-cyclodextrin bimodal conjugate: nanosized drug transporter and potent phototoxin[END_REF] owing to the presence of three phenyl groups in the molecular structure suitable for cavity inclusion.

Gemcitabine (GEM, 2',2'-difluoro-2'deoxycytidine, dFdC), is a pyrimidine nucleoside analogue anticancer prodrug used in the treatment of a range of solid tumors, also a broad spectrum antiviral agent [START_REF] Lee | Gemcitabine, a broad-spectrum antiviral drug, suppresses enterovirus infections through innate immunity induced by the inhibition of pyrimidine biosynthesis and nucleotide depletion[END_REF]) and a potent radiosensitizer [START_REF] Vanderveken | Gemcitabine-based chemoradiation in the treatment of locally advanced head and neck cancer: systematic review of literature and meta-analysis[END_REF]. GEM does not carry groups suitable for the cavity of βCD, and binds βCD weakly (46 ± 2 M -1 ), as we derived from NMR titrations (Fig. B19). Since the adamantyl group (ADA) is typically considered as an ideally sized moiety for strong βCD inclusion (10 4 -10 5 M -1 , [START_REF] Rekharsky | Complexation thermodynamics of cyclodextrins[END_REF], it became clear that the best approach would be to employ a hydrolysable ADA derivative of GEM in the studies, as needed, and therefore 4-N-(1-adamantoyl)-2',2'-difluoro-2'-deoxycytidine (GEMADA) was prepared (Scheme A2).

DLS experiments.

Solutions of the pre-assembled NPs were co-incubated with model guest 1adamantylcarboxylic acid (ADACOOH), TamCit and GEM (Table 2, Fig. 2E). Incubation with ADACOOH results in doubling (TPPOCNHbmC2/C5) and tripling (TPPOCNHbmC11) of the NP diameters, reaching ~ 200 nm. On the contrary, the aggregates of the parent porphyrins do not seem to be particularly disturbed (Fig. 2F). Consequently, the NPs seem to maintain a number of available cavities and the entering guests push the linker chains away causing the NPs to expand. Incubation with the very poor guest, GEM, causes moderate growth of NPs, suggesting that non-cavity entanglement can be also a loading mechanism. TamCit causes the NPs to grow considerably (TamCit), exceeding 100 nm for the conjugate with C5 and C11 linkers (Table 2), suggesting both cavity encapsulation and entanglement in the NPs. Table B4 lists λmax shfits in the presence of guests. Overall, NPs seem to grow with the length of the chain between βCD and TPP moieties, remaining below 200 nm thus still considered suitable for cell studies [START_REF] Dolai | Nanoparticle size effects in biomedical applications[END_REF][START_REF] Oh | Endocytosis and exocytosis of nanoparticles in mammalian cells[END_REF]. The PDI values remained reasonably low, ranging from 0.14 to 0.29 nm.

Inclusion studies by NMR. Direct proof of guest encapsulation in the βCD cavity of

TPPOCNHbmCx was provided by NMR spectroscopy using pMβCD to cap, isolate and solubilize the TPP part. The spectra confirm strong and specific TPP moiety inclusion in pMβCD (Fig. B20A-D, Fig. B21). Table B3 lists the stoichiometries and binding constants (K ~ 10 11 M -2 , Fig. B22) of pMβCD/conjugate inclusion complexes. The tight pMβCD/TPPOCNHbmCx complexes form additional complexes with TamCit via the available βCD moiety (Fig. B21A, Fig. B21B-D).

UV-Vis spectral analysis.

Analysis of the absorption spectra in DMF and in PBS of the NPs alone and drug-loaded (Fig. B23) was performed with 2 nd derivatives (Fig. B24). In DMF all conjugates display a single sharp peak at the same λmax = 418.5 nm (Fig. B24A) confirming the presence of monomers only. In contrast, the conjugates reveal rather broadened and both red-and blue-shifted Soret bands in PBS (Fig. B24B-D): those at ~ 403 nm suggest formation of H-aggregates while the redshifted ones at ~ 422 nm suggest formation of J-aggregates [START_REF] Maiti | J-and H-aggregates of porphyrin-surfactant complexes: time-resolved fluorescence and other spectroscopic studies[END_REF][START_REF] Villari | Spontaneous self-assembly of water-soluble porphyrins having poly(ethylene glycol) as branches: dependence of aggregate properties from the building block architecture[END_REF][START_REF] Pescitelli | Application of electronic circular dichroism in the study of supramolecular systems[END_REF]. Upon addition of the drugs, the intensities of both bands change suggesting redistribution of the respective populations while bathochromic shifts and further broadening reveal the enhancement of J-aggregates and the enlargement of the NPs, in agreement with the DLS results.

Theoretical calculations, as in section 2.3.3, show that entry of ADACOOH in the cavity of TPPOCNHbmC11 untwists the aliphatic chain, resulting in extension of the conjugate from 2.9 nm to 3.6 nm (Fig. B24E).

Photostability and 1 O2 production

The decrease in absorption intensity of the Soret bands in the UV-Vis spectra (Fig. B25A-C) was monitored upon irradiation at λ = 630 ± 10 nm, 0-28.34 J/cm 2 corresponding to 0-120 min, in PBS.

At ~4 J/cm 2 , used for irradiation of the cells (section 2.8) the photobleaching of all conjugates is only ~ 10% reaching ~30% at the end (Fig. B25D). The similar behavior is attributed to the common assembly mode of NPs in the aqueous medium. Moreover, irradiation of the conjugates in PBS in the presence of 9,10-anthracenediylbis(methylene)dimalonic acid (ABDA) confirmed the generation of 1 O2 by all conjugates (Fig. B26), even in as NPs. ABDA is known to react instantly with 1 O2 to form an endoperoxide product thus resulting in its photobleaching [START_REF] Entradas | The detection sensitivity of commonly used singlet oxygen probes in aqueous environments[END_REF][START_REF] Song | Phytoalexin phenalenone derivatives inactivate mosquito larvae and root-knot nematode as type-II photosensitizer[END_REF]. The rate constants of 1 O2 production for TPPOCNHbmC5/C2 (~10 x 10 -4 M -1 ) are similar to or greater than that of the known PS meso-5,10,5,20-(3-hydroxyphenyl)porphyrin (mTHPP) (~ 6.6 x 10 -4 M -1 , Table B5) thus indeed, the NPs are photoactive.

Confocal Laser Scanning Microscopy

Live imaging of human breast adenocarcinoma MCF-7 cells incubated for 24 h with NPs of TPPOCNHbmC5 (Fig. 3A1) and TPPOCNHbmC2 (Figs. supporting localization of the conjugates in the lysosomes. Mander's Coefficients (M1 and M2) [START_REF] Manders | Measurement of co-localization of objects in dual-colour confocal images[END_REF][START_REF] Pike | Quantifying receptor trafficking and colocalization with confocal microscopy[END_REF] were calculated and M1 (fraction of red fluorescence, i.e. TPPOCNHbmC2 in areas with green fluorescence, i.e. LysoTracker) was found >0.80 (Fig. B28E) proving the exceptional selectivity of TPPOCNHbmC2 NPs in targeting the lysosomes. Further confirmation of FRET was obtained in solution: upon addition of TPPOCNHbmC2 gradual quenching of the lysotracker emission band at 510 nm solution was registered, either using λex = 488 (Fig. 3C) or 470 nm (Fig. B29C). Concomitant increase of the bands at 654 and 716 nm due to the emission of the porphyrin moiety was noted. The rate of quenching was nearly identical at both λex settings (Fig. B29D) and there was no TPPOCNHbmC2 emission (Fig. B29E). The LysoTracker used is a BODIPY derivative that when in lysosomes, can presumably enter available βCD cavities of co-localized conjugates keeping the chromophores at FRET distance. Similar effects have been recently reported in cells between a BODIPY-pMβCD conjugate (donor) and an included porphyrin (acceptor) [START_REF] Nakagami | FRET-based in-cell detection of highly selective supramolecular complexes of meso tetraarylporphyrin with peptide/BODIPY-modified per-Omethyl-β-cyclodextrins[END_REF].

The self-assembled NPs are apparently taken up by the cells via endocytosis thus they end up in vesicles within the cells. The very bright fluorescence intensity in the images suggests that it is possible to find monomeric conjugates anchored in the organelle membranes, thus model liposomes were employed as mimics of subcellular compartments to investigate such a possibility.

Liposomes as models of vesicular organelles-incorporation of the conjugates in the membranes

1,2-Dioleoyl-sn-glycero-3-phosphocholine (DOPC) liposomes were prepared by the Bangham method [START_REF] Bangham | Diffusion of univalent ions across the lamellae of swollen phospholipids[END_REF] in the presence of TPPOCNHbmC5 ,TPPOCNHbmC11 or TPP.

The incorporation efficiency of the conjugates was very high (> 99%). The size, size distribution, zeta potential, and morphology of the resulting liposomes were indicative of stable and well-formed entities (Table B6, Fig. B30). Comparison of the UV-Vis absorption of the DOPC/conjugates liposomes in water with those of the respective conjugates in DMF (Fig. B31), where the conjugates are presumably monomeric, reveals complete similarity regarding both λmax and band shape. This remarkable result suggests that the conjugates have been incorporated in the liposome membranes as monomers and the absorption bands do not arise from NPs enclosed in the liposome aqueous interior, where they are expected to aggregate and give rise to much broadened bands. Additionally, overnight co-incubation of blank DOPC liposomes with a 2 μM PBS solution of pre-assembled porphyrin-βCD NPs resulted in increased absorption intensity and decreased broadness of the Soret band for TPPOCNHbmC2/C5 NPs (Figs. B32A,B,D), suggestive of their effective interaction with the liposome membranes. In contrast, TPPOCNHbmC11 displayed slightly greater broadness of the Soret band (Fig. B32C) probably due to the prevailing enlargement of the NP size with time (Fig. B17C3).

Using a liposome irradiation setup (~50 mW/cm², 300-400 nm centered at 350 nm) the successful production of ROS by TPPOCNHbmC5 in DMF was confirmed using the ROS scavenger methyl linoleate, (Fig. B33). The DOPC/TPP/CF liposomes loaded with carboxyfluorescein (CF) as model cargo showed clear release of CF with irradiation time (Fig. B34A), demonstrating that the integrity of the liposome membrane has been compromised due to the ROS generated by TPP. Surprisingly, the release profile of CF from the formulation DOPC/TPPOCNHbmC5/CF (Fig. B34B) was practically that of the blank liposomes loaded with CF. Excitation of a CF solution in PBS at λexc = 470 nm (Massu et al., 2000) and gradual increase the amounts of TPPOCNHbmC5 added (no absorption at 470 nm) revealed decreased CF emission (Fig. B34C). The emission spectrum of CF (λem = 480 -600 nm), overlaps with the absorption region of TPPOCNHbmC5 (350 -680 nm, Fig. 1) and FRET between the CF donor and the porphyrin moiety acceptor in TPPOCNHbmC5 mediated by the βCD cavity can result in decreased CF emission and emergence of TPPOCNHbmC5 fluorescence (653 and 715 nm, Fig. B34C). Indeed, the 2D ROESY NMR spectrum of βCD/CF confirmed very strong binding (Fig. B34D) undisturbed by the addition of the competitive guest ADACOOH.

The liposome experiments show that the conjugates, even as assembled NPs, can be incorporated in the bilayers as monomers. Moreover, guest release from the interior of the liposomes can be greatly affected by the formation of strong inclusion complexes with available βCD cavities. The incell FRET between TPPOCNHbmC2 and LysoTracker described previously (Fig. 3) suggests that by analogy, molecules entrapped in the lysosomes can also form inclusion complexes with the available cavities of the membrane-embedded conjugates.

8. Cytotoxicity and photokillinga synergistic PCI effect

MCF-7 cells were incubated in 96-well plates for 24 h with either the pre-assembled NPs alone or with the drug-loaded NPs (with TamCit and GEMADA) or with the drugs alone. The appropriate sample groups were then irradiated at 640 nm with a custom-made LED array lamp at light-doses suitable to achieve LD50 or the highest synergistic effect (4.18 J/cm 2 , Figs. B35A-C). Irradiation of the untreated cells confirmed that light alone does not induce toxicity (Fig. B35D). Subsequently, all plates including dark, media only, and drug only controls, were evaluated for cytotoxicity with the MTT assay at the 24 h and 48 h time-points (Fig. 4).

The dark experiments of Fig. 4A reveal that TamCit and GEMADA alone confer low toxicity on the cells at 24 h (< 20% cell death), compared to dark control, and have similar effects after 48 h.

The conjugates alone at the selected concentrations (Figs. 4B-D) are devoid of toxicity in the dark at 24 h and the cells seem to recover with time (48 h). Similarly, the cells with the NPs loaded with either TamCit or GEMADA largely retain their viability at 24 h and even proliferate at 48 h, demonstrating lack of dark toxicity, despite the chemotoxic payload (Fig. 4B-D).

Upon irradiation, the drugs alone at 24 h and 48 h (Fig. 4A) confer little damage, and the viability of the cells is retained at ~80% with a tendency to recover with time. On the contrary, the conjugates TPPOCNHbmC2 (Fig. 4B) and TPPOCNHbmC5 (Fig. 4C) elicit considerable toxicity as only ~30% of the cells remain viable at 24 h, displaying strong phototoxic (PDT) effects. Nevertheless, the cells proliferate after additional 24 h (~60 % viability) indicating that at the given set of experimental conditions the phototoxicity effect is temporary. In sharp contrast, the TPPOCNHbmC2 and TPPOCNHbmC5 NPs loaded with either TamCit or GEMADA not only confer stronger PDT effects on the cells at 24 h (~15-20% viability) but they also halt the proliferation of the cells for the next 24 h suggesting action of the drugs and manifestation of PCI.

Under the same conditions, cells incubated with TPPOCNHbmC11 NPs alone or loaded with the drugs, show poor PDT effects (~70% viability at 24 h) and questionable PCI effects (Fig. 4D), evidently due to low uptake (Fig. 3A3). The unloaded conjugates' phototoxicity compares sufficient well with that of mTHPP used as a positive control (Fig. B36) which, however, has very different localization preferences in the cells. Different localization locci had also been observed in our previously reported water soluble conjugates (Aggelidou et al. 1013), which were also less efficient regarding PDT than the current, insoluble amphiphilic systems. The results with the drug-loaded TPPOCNHbmC2/C5 NPs show strong synergy between the photodynamic and chemotherapeutic effects (Table B7): the values of the calculated synergy index, α, [START_REF] Christie | Synergistic chemotherapy by combined moderate hyperthermia and photochemical internalization[END_REF], Appendix A) are ~1.5 at 24 h and ~3 at 48 h. This outcome can be convincingly explained by 1 O2 photogeneration that compromises lysosome membranes and subsequently induces drug release (PCI). Strong synergy is observed not only with TamCit, a drug suitable for treatment of the estrogen-receptor β-positive MCF-7 cell line, but also with GEMADA. The first line anticancer drug GEM requires nucleoside transporters for cell uptake [START_REF] Mackey | Gemcitabine transport in xenopus oocytes expressing recombinant plasma membrane mammalian nucleoside transporters[END_REF]. When intracellularly, GEM undergoes sequential phosphorylation steps to attain the active form that inhibits both DNA synthesis and the enzymes of nucleotide metabolism of cancer cells [START_REF] Baker | 2'-Deoxy-2'-methylenecytidine and 2'-deoxy-2',2'-difluorocytidine 5'-diphosphates: potent mechanism-based inhibitors of ribonucleotide reductase[END_REF][START_REF] Mini | Cellular pharmacology of gemcitabine[END_REF]. In our comparison experiments, the cell viability using GEM alone is found to decrease to ~60% after either 24 h or 48 h incubation, with or without irradiation (Fig. B37) suggesting low uptake. GEMADA alone, under the same conditions, appears to be even less toxic than GEM (Fig. B36) as cell viability only drops to ~80%. On the contrary, under irradiation conditions, the cells treated with GEMADA-loaded NPs suffer profound reduction of their viability (Figs. 4B-C), as discussed above. We can postulate, therefore, that due to the presence of the adamantyl group GEMADA binds strongly with the βCD cavities of the NPs and is endocytosed along with them, thus bypassing considerably its difficulty for uptake and potentially minimizing off-target effects. Under irradiation the drug is released to exert its toxic effects on-site. Various GEM modifications to overcome current drawbacks and improve its clinical efficacy have been reported [START_REF] Moysan | Gemcitabine versus modified gemcitabine: a review of several promising chemical modifications[END_REF]. Here, the adamantyl group is bonded to the 4-(N)-position of GEM (Scheme A2) leaving the -OH site free for phosphorylation and possibly also protecting the molecule against immediate deamination by cytidine deaminase, a process considered responsible for resistance to the drug. Our approach to GEM modification may prove suitable for other drugs of the nucleoside analogue family for delivery via similar nano-platforms.

Conjugate Conjugate +TamCit

Conjugate

The aggregating properties of glycosylated or "sugar-coated" porphyrinoids have been recognized, investigated and assessed and their importance in the diagnostic and therapeutic outcome has been highlighted [START_REF] Singh | Glycosylated porphyrins, phthalocyanines, and other porphyrinoids for diagnostics and therapeutics[END_REF]. Many parameters contribute to the size and stability of the aggregates that affect both the photophysical properties and cell uptake. The results presented herein demonstrate that uncomplicated porphyrin-βCD conjugates form suitable NPs and that loading with small molecular weight chemotherapeutic drugs or tailored pro-drugs, is feasible.

Interaction with cell membrane components is expected to destabilize the NPs, however, it is also expected that even <60 nm entities would be large enough (MW ~2 kDa) to become endocytosed and end up in endosomes and lysosomes. Further, the fact that upon irradiation GEM alone confers little damage to the cells and GEMADA alone even less, but the GEMADA/conjugate formulation becomes highly toxic, highlights a method to bypass natural cell uptake mechanisms and direct the drug together with the conjugate into the lysosomes for PCI. Our present NPs, completely devoid of dark toxicity, can be enlisted as a promising new set of biocompatible systems and can be considered, in addition to other nano-assemblies [START_REF] Xue | Porphyrin-based nanomedicines for cancer treatment[END_REF], as efficient vehicles for the photodelivery of small drugs.

Conclusions

In a few synthetic steps we have prepared three conjugates of βCD with monocarboxy-methyl-, -

  Scheme 1. Molecular structure of the porphyrin-βCD conjugates, TPPOCNHbmCx, and % yields of their synthesis from porphyrins TPPOCOOHCx and mono(6-amino-6-deoxy)-βCD (bmNH2).

Fig. 1 .

 1 Fig. 1. UV-Vis absorption spectra (2 μΜ solutions): A) TPPOCNHbmCx, DMF; B) TPPOCOOHCx, DMF; C) TPPOCNHbmCx, PBS; D) TPPOCOOHCx, PBS. Emission spectra (2 μΜ solutions, A = 0.02, λexc = 515 nm) of: E) TPPOCNHbmC2; F) TPPOCNHbmC5; G) TPPOCNHbmC11 in 1: (---) DMF; 2: (---) PBS; 3:

FFig. 2 .

 2 Fig. 2. (A) TEM image of TPPOCNHbmC11 NPs at 20 μM (without contrasting agent); (B-D) Number (%) vs size DLS profiles of (B) TPPOCNHbmC2, (C) TPPOCNHbmC5 and (D) TPPOCNHbmC11 NPs at 2, 5, 10 and 20 μΜ concentrations in PBS*. DLS profiles of number (%) vs size of (E) TPPOCNHbmCx in comparison with (F) TPPOCOOHCx (2 μM in PBS) and in the presence of guest molecules (GEM.HCl, TamCit, ADACOOH). *PBS is with 4% DMSO (v/v), pH 7.4

  2.3.2. Dynamic light scattering (DLS). The pre-assembled conjugates in the range 2 to 20 μΜ behave uniformly (Fig. 2B-D, Figs. B17C1,C2) and form ~ 60 nm NPs with polydispersity indices (PDI) ≤0.22 (entries 1-3, Table

  3A2) using λex = 514 nm and λem = 637 -670 nm, revealed abundant and very intense punctate red fluorescence, suggestive of high uptake, in contrast to TPPOCNHbmC11 (Figs. 3A3, B27) that is not efficiently internalized. The dotted fluorescent patterns observed indicate that the conjugates are localized at numerous intracellular vesicular organelles such as endosomes and lysosomes. There are no indications of either cytoplasm or nuclear membrane localization. Brief co-incubation of the TPPOCNHbmC2-treated cells with LysoTracker® Green DND-26 (λex = 488 nm, λem = 501 -538 nm) resulted in multiple yellow dotted areas in the overlaid images (Figs. 3B1-B3, B28),

  Fig. 3. (A-B): Confocal microscopy images of MCF-7 cells incubated for 24 h with NPs of conjugates (10 μΜ): (A1-3, B1) images with λex = 514 nm, laser intensity 3% and λem = 637 -670 nm: (A1) TPPOCNHbmC2; (A2) TPPOCNHbmC5; (A3) TPPOCNHbmC11; B1) TPPOCNHbmC2 NPs treated with LysoTracker® Green DND-26 (0.3 μM); (B2) cells of B1 imaged with λex = 488 nm, laser intensity 1%, λem = 501 -538 nm; (B3) overlay of images B1 and B2 with yellow spotted areas indicating high co-localization; (B4) cells in B1 imaged with λex = 488 nm and laser intensity 3% but detected with λem = 637 -670 nm indicates FRET between the lysotracker and the porphyrin moiety of TPPOCNHbmC2; (C) Decreased emission at λem = 510 nm (green arrow) of a LysoTracker® Green DND-26 solution (0.3 μM) in the presence of increased amounts of TPPOCNHbmC2 (0-12 μM) and emergence of TPPOCNHbmC2 fluorescence at λem = 625 -760 nm (red arrow) registered following λexc = 488 nm in PBS* indicate FRET between the two molecules.. * PBS is with 4% DMSO (v/v), pH 7.4.

Fig. 4 .

 4 Fig. 4. Viability of MCF-7 cells following 24 h incubation with porphyrin-βCD NPs (10 μM) without or with anticancer drugs (7.5 μM), without (white and light grey bars) or with irradiation at 4.18 J/cm 2 , λ = 640 nm (dark grey and black bars). The MTT assays were performed at 24h (dark grey bars) or 48 h (black bars) postirradiation: (A) Control experiments with cells alone (control), TamCit or GEMADA; (B) TPPOCNHbmC2; 425 (C) TPPOCNHbmC5; (D) TPPOCNHbmC11, alone and loaded with the drugs. Error bars are shown in each case.

  butyl and -decyl-TPP. The conjugates show similar photophysical properties both in DMF and in aqueous media, regardless of the different length of the aliphatic chain linkers between the TPP and βCD moieties. Importantly, the conjugates self-assemble into very similarly sized, photoactive NPs that specifically target and accumulate in the lysosomes of MCF-7 cells. Liposome experiments show that the NPs can interact with lipid membranes wherein they can be lodged as monomers. We demonstrate high overall photo and chemo-toxicities following irradiation of cells incubated with NPs loaded with TamCit or GEMADA due to significant synergy developed that suggests operation of PCI, the effects lasting for at least 48 h. Dark experiments demonstrate complete lack of toxicity of the NPs, either empty or drug-loaded, thus the drugs are released only with the action of light. Moreover, attachment of an adamantyl group can direct GEM along with the NPs to endocytic vesicles, enabling use of this potent small drug in PCI treatments. The radiosensitizing properties of GEM also point to the possibility that the NPs could be interesting vehicles for combined chemo-, photo-and radio-treatment of tumors.CRediT authorship contribution statementS.P.: Investigation, Methodology, Analysis, Writing & Editing; B.M. and A.A.: Methodology, Analysis & Editing; L. B.-M. Investigation, Analysis, & Writing; T.R.: Methodology & Analysis; A.R.R.: Investigation & Analysis; N.B.: Methodology & Review; G. C.: Methodology & Editing;

Table 1

 1 Emission

	Entry	Compound	Lifetime (

lifetimes (τ) of the porphyrin-βCD conjugates and parent porphyrins alone and with pMβCD, in DMF i and in PBS ii , λexc = 515 nm, 2 μM, 25 ºC.

ns) DMF Lifetime (ns) PBS Lifetime (ns) +pMβCD, PBS τ1

				(%)	τ2 (%)	τ1 (%)	τ2 (%)
	1 TPPOCNHbmC2	9.1	1.3 (44.1) 4.6 (55.9)	2.6 (5.9)	14.5 (94.1)
	2 TPPOCNHbmC5	9.9	0.8 (47.8) 4.5 (52.2)	1.4 (1.9)	12.6 (98.1)
	3 TPPOCNHbmC11	10.7	1.2 (50.4) 4.8 (49.6)	1.8 (21.1)	6.6 (78.9)
	4	TPPOCOOHC2 i	11.2	1.3 (19.4) 5.3 (80.6)	12.0 (100)	-
	5	TPPOCOOHC5 i	10.9	0.7 (69.5) 3.7 (30.5)	0.7 (7.8)	11.1 (92.2)
	6 TPPOCOOHC11 i	11.2	0.8 (97.7) 2.7 (2.3)	1.0 (94.7)	5.9 (5.3)

i

Panagiotakis et al., 2022, ii

Phosphate buffer saline containing 4% DMSO (v/v), pH 7.4. 9

Table 2

 2 DLS measurements of self-assembled conjugates and the corresponding porphyrins, 2 μM in PBS i and in the presence of drugs.

	#	Compound ii		Size (nm) (PDI iii )
			NP	+ TamCit iv	+ GEM v	+ ADACOOH vi
	1	TPPOCNHbmC2	57 ± 12 (0.22)	90 ± 7 (0.25)	71 ± 5 (0.23)	100 ± 6 (0.23)
	2	TPPOCNHbmC5	60 ± 3 (0.17)	121 ± 19 (0.24)	102 ± 4 (0.29)	125 ± 10 (0.27)
	3	TPPOCNHbmC11	60 ± 4 (0.19)	146 ± 16 (0.23)	119 ± 25 (0.23)	190 ± 31 (0.14)
	4	TPPOCOOHC2	88± 14 (0.39)	-	-	64± 40 (0.69)
	5	TPPOCOOHC5	79± 12 (0.81)	-	-	87± 20 (0.91)
	6	TPPOCOOHC11	105± 37 (0.79)	-	-	101± 44 (0.84)
	 i Phosphate buffer saline containing 4% DMSO (v/v), pH 7.4, r.t; ii Measured 24 h after preparation of the solutions.

A.G.C. Resources & Review; M. G.: Methodology, Analysis; T.A.T.: Resources, Analysis, Review; K. B.: Resources, Analysis & Review; C. L.: Resources, Analysis, Review; M. P.: Resources, Analysis, Review; K.Y.: Conceptualisation, Investigation, Analysis, Writing, Review and Editing, Acquisition of funds, Resources, Coordination. Acknowledgments Funding Funding by the General Secretariat for Research and Technology (Greece) in the frame of the EuronanoMed II network for the project PCInano (T3EPA-0048) and infrastructure support by the project "National Infrastructure in Nanotechnology, Advanced Materials and Micro-/ Nanoelectronics" INNOVATION EL (MIS 5002772) funded by the Operational Programme "Competitiveness, Entrepreneurship and Innovation" (NSRF 2014-2020) and co-financed by Greece and the European Union are gratefully acknowledged. Appendix A and Appendix B. Experimental and Supplementary data

Declaration of competing interest

There are no conflicts to declare.