Arnaud Beddok
email: arnaud.beddok@curie.fr

Caroline Saint-Martin

Samar Krhili

Catherine Ala Eddine

Laurence Champion

Anne Chilles

Farid Goudjil

Sofia Zefkili

Malika Amessis

Dominique Peurien

Ludivine Catteau

Olivier Choussy

Christophe Le Tourneau

Jerzy Klijanienko

Remi Dendale

Irene Buvat

Gilles Créhange

Valentin Calugaru

Curative high-dose reirradiation for patients with recurrent head and neck squamous cell carcinoma using IMRT or proton therapy: Outcomes and analysis of patterns of failure

Keywords: Reirradiation, head and neck carcinoma, IMRT, proton therapy, toxicity

Introduction

Radiation therapy (RT), either definitive or after surgery, is a cornerstone of local treatment of head and neck cancer (HNC), with rates ranging from 77% to 91%, depending on the location of the tumor. [START_REF] Petit | Chemotherapy and radiotherapy in locally advanced head and neck cancer: an individual patient data network meta-analysis[END_REF] Several long-term clinical trials have shown that local and/or regional recurrence (LR) rates can be as high as 30%. [START_REF] Setton | Intensity-Modulated Radiotherapy in the Treatment of Oropharyngeal Cancer: An Update of the Memorial Sloan-Kettering Cancer Center Experience[END_REF]3 Surgery is the treatment modality used for this type of recurrence when possible. [START_REF] Nichols | Surgical salvage of the oropharynx after failure of organ-sparing therapy[END_REF][START_REF] Bachar | Long-term outcome analysis after surgical salvage for recurrent tonsil carcinoma following radical radiotherapy[END_REF][START_REF] Taussky | Salvage surgery after radical accelerated radiotherapy with concomitant boost technique for head and neck carcinomas[END_REF][START_REF] Wong | Salvage of recurrent head and neck squamous cell carcinoma after primary curative surgery[END_REF] According to a meta-analysis of 32 studies with a total of 1080 patients, a survival rate of 39% can be expected at 5 years after salvage surgery. [START_REF] Goodwin | Salvage Surgery for Patients With Recurrent Squamous Cell Carcinoma of the Upper Aerodigestive Tract: When Do the Ends Justify the Means?[END_REF] Postoperative reirradiation (reRT) allowed significant improvements in locoregional failure-free survival (LFFS) and disease-free survival (DFS). [START_REF] Janot | Randomized Trial of Postoperative Reirradiation Combined With Chemotherapy After Salvage Surgery Compared With Salvage Surgery Alone in Head and Neck Carcinoma[END_REF] Exclusive reRT with palliative or curative intent is also a possible option, particularly when the patient is inoperable or the tumor cannot be removed. [START_REF] Tortochaux | Randomized phase III trial (GORTEC 98-03) comparing re-irradiation plus chemotherapy versus methotrexate in patients with recurrent or a second primary head and neck squamous cell carcinoma, treated with a palliative intent[END_REF][START_REF] Spencer | Final report of RTOG 9610, a multi-institutional trial of reirradiation and chemotherapy for unresectable recurrent squamous cell carcinoma of the head and neck[END_REF] Nevertheless, the local relapse rate, especially in the irradiated field, remains high. [START_REF] Popovtzer | The pattern of failure after reirradiation of recurrent squamous cell head and neck cancer: implications for defining the targets[END_REF][START_REF] Takiar | Reirradiation of Head and Neck Cancers With Intensity Modulated Radiation Therapy: Outcomes and Analyses[END_REF] Moreover, several important trials have shown that reRT for HNC can result in up to 8% treatment-related deaths. [START_REF] Spencer | Final report of RTOG 9610, a multi-institutional trial of reirradiation and chemotherapy for unresectable recurrent squamous cell carcinoma of the head and neck[END_REF][START_REF] Langer | Phase II Study of Low-Dose Paclitaxel and Cisplatin in Combination With Split-Course Concomitant Twice-Daily Reirradiation in Recurrent Squamous Cell Carcinoma of the Head and Neck: Results of Radiation Therapy Oncology Group Protocol[END_REF] In other more recent studies, up to 35% of patients reirradiated with intensity-modulated radiation therapy (IMRT) developed grade 3 toxicities. [START_REF] Takiar | Reirradiation of Head and Neck Cancers With Intensity Modulated Radiation Therapy: Outcomes and Analyses[END_REF][START_REF] Duprez | High-dose reirradiation with intensity-modulated radiotherapy for recurrent head-and-neck cancer: Disease control, survival and toxicity[END_REF] Several techniques, including proton therapy (PT), [START_REF] Romesser | Proton beam radiation therapy results in significantly reduced toxicity compared with intensity-modulated radiation therapy for head and neck tumors that require ipsilateral radiation[END_REF] stereotactic RT, [START_REF] Phan | Reirradiation of Head and Neck Cancers With Proton Therapy: Outcomes and Analyses[END_REF] and brachytherapy [START_REF] Dionisi | Clinical results of proton therapy reirradiation for recurrent nasopharyngeal carcinoma[END_REF] could be useful to decrease this risk of frequent and severe toxicities. Our objectives were to analyze management, outcomes and toxicities, including patterns of failure, in patients treated with curative reRT (either by intensity-modulated radiation therapy [IMRT], or by proton therapy [PT]) for recurrent head and neck squamous cell carcinoma (HNSCC).

Material and Methods

Patient population

Among the 55 patients reirradiated with IMRT or PT for recurrent HNC at Institut Curie, 23 suffered from HNSCC. The reRT was delivered after surgery when complete resection was not obtained or without surgery when lesions appeared to be unresectable. The 11 patients in the present series reirradiated with PT had been referred from seven separate institutions and practices to our proton therapy center, most often because additional photon therapy was not deemed feasible. For the patients evaluated at our academic center, the selection of PT as the reRT modality was decided when the dosimetric gains of PT were believed advantageous because photon-based reRT could not adequately cover the reRT target without exceeding the critical normal tissue constraints, or would result in an excessive risk of toxicity. This was most often related to the location of the recurrent disease with the disease adjacent to or involving the skull base. The diagnosis of local relapse had been proven by biopsy or surgical specimen. The initial staging evaluation included clinical examination and magnetic resonance imaging (MRI) or contrast-computed tomography (CT) of the neck, and positron emission tomography (PET) for distant disease assessment. The treatment of each patient was discussed by a multidisciplinary team. This study was approved by the Ethics Committee of the Institut Curie on 7 July 2020 (DATA200213).

Radiation Therapy

All patients underwent simulation for radiation treatment planning purposes, which consisted of the fabrication of a customized thermoplastic mask and a thermoformed mattress for immobilization in the supine position, followed by CT imaging without and with contrast enhancement. The target volume and normal tissue structures were delineated on each axial CT slice, supplemented with fused diagnostic MRI, and/or PET-CT scans. TomoTherapy Hi-Art Treatment System (Accuray, Sunnyvale, CA) or the Eclipse photon therapy treatment planning system (Varian, Palo Alto, CA) was used for the IMRT plans, and the ISOgray (Dosisoft, Cachant, France) or Eclipse proton treatment planning system (Varian, Palo Alto, CA) for PT plans. For patients without upfront salvage surgery, the high-risk CTV (HR-CTV) included the GTV of recurrence with an isocentric margin of 5 mm.

For patients irradiated after salvage surgery, the HR-CTV included the tumor bed and involved nodal stations, while the low-risk CTV (LR-CTV) included the nodal stations above and below the involved nodal areas. The dose to the HR-CTV was 59.4-70.2 Gy (1.8-2 Gy/fraction), and the dose to the LR-CTV was 56-59.4 Gy (1.8 Gy/fraction). Patients in the IMRT group received helicoidal IMRT (Tomotherapy), using a simultaneous integrated boost (SIB). For patients treated with PT, the target volumes were treated with 201 MeV proton beams using the passive scattered proton therapy (PSPT) technique (Double Scattering Beam). The RBE used for PT was 1.1. Figure 1 illustrates the plan for IMRT and PSPT reirradiation.

Follow-up

Each follow-up visit included a clinical physical examination. An MRI, CT, and/or PET-CT of the neck was conducted every 6 months during the first 5 years, and every 12 months thereafter or until death. Locoregional failure (LRF) was detected within the head and neck by either clinical examination, imaging, and/or biopsy. The Common Terminology Criteria for Adverse Events, Version 5.0 (CTCAE v5.0), formerly called the Common Toxicity Criteria (CTC or NCI-CTC), were used to assess the late toxicities of RT at each patient visit. For patients with evidence of local-regional recurrence or distant metastasis, additional clinical or imaging examinations were performed to confirm or rule out disease progression at the discretion of the treating physician.

Patterns of failure study

For this section, we used the same approach already applied in three previously published studies.12, 19, 20 For all patients with locoregional relapse or second primary HNSCC after reRT, the recurrent tumor volume (Vrecur) was identified on MRI and/or PET scans obtained at the time of diagnosis of recurrence. The contours of this volume were validated for each patient by at least a nuclear physician (LC) and/or a radiologist (CAE) expert in this pathology. The exact site and extent of each tumor were then compared visually to the pretreatment planning CT datasets, focusing on the 95% isodose lines. The recurrences were categorized according to previously published criteria as occurring inside or outside the previously irradiated targets: the Vrecur was deemed "in-field," if the majority of Vrecur was within the 95% isodose; "marginal," if ≤50% of Vrecur was within the 95% isodose; or "outside," if <20% of the Vrecur was inside the 95% isodose (Figure 2).

Statistical Analysis

Follow-up was calculated from the date of the end of reRT to the last clinical follow-up. The median follow-up was estimated by the inverted Kaplan Meier method. Baseline characteristics were summarized as numbers and percentages for qualitative data, and as means and standard deviations or medians with the minimum and maximum (or inter-quartile range) for continuous variables. The Chi 2 test or Fisher's exact test was used for the analysis of the contingency tables. Overall survival (OS) was defined as the time between the date of the end of reRT and the date of death for deceased patients. Patients still alive were censored at the date of their last news. Locoregional failure-free survival (LFFS), including local and nodal progression, was calculated from the date of the end of reRT until the date of LRF. Disease-free survival (DFS) was calculated as the time between the date of the end of reirradiation and the first event: locoregional or distant relapse or death. In the absence of any event, patients were censored at the date of their last news. Survival distributions were estimated by the Kaplan-Meier method and compared using the log-rank test. Univariate Cox regression models were used to assess the relative influence of prognostic factors. The added value of each variable to the Cox model was determined using a likelihood ratio test. As death is a competing risk for locoregional recurrence, the risk of locoregional recurrence based on the survival analysis was assessed according to methodological recommendations within the framework of competing risks, using cumulative incidence functions and models for the sub-distribution hazards ratio (SHR). A Fine-Gray model was implemented to illustrate the regression models of SHRs. A p value <0.05 was considered statistically significant. Analyses were carried out using software R 3.6.3.21

Results

Patient characteristics

The 23 patients analyzed in this study had HNSCC, including 11 (47.8%) with the undifferentiated carcinoma of nasopharyngeal type (UCNT, World Health Organization type III). Baseline characteristics are summarized in Table 1. The median age of the cohort was 47.1 years old. The main initial disease sites were nasopharynx (N = 14). The main sites of recurrence were the nasopharynx (N = 7), lymph nodes (N = 5) and the oropharynx (N = 5). Recurrences were essentially locoregional and staged III-IVa (N = 16). Nine patients underwent surgery prior to reRT, seven had neoadjuvant chemotherapy (most often combining cisplatin and docetaxel), and six had concomitant chemoradiotherapy (most often cetuximab or cisplatin, Table 2).

Reirradiation treatment details

Reirradiation treatment details are summarized in Table 2. The median time between the two irradiations was 29.3 months (IQR: 15.3-37.2 months). The median maximum dose prescribed to the GTV was 66 Gy (IQR: 60.5-66.6 Gy), median overall treatment time: 54 days (IQR: 48.5-56.5 days), median CTV and PTV volumes: 49.8 cc (IQR: 32.4-88.8 cc) and 124.3 cc (IQR: 76.9-208.6 cc), respectively. Half of the patients received PT (especially recurrent UCNT patients) and the other half received IMRT (Supplementary Table 1). The cumulative dose to the organs at risk (OAR) greatly exceeded the dose usually tolerated for a first irradiation (Table 2). The OAR dose constraints for reRT were chosen by assuming an empirical "forgetting factor" of 5% per year for nerve structures. 22 For other structures, the rule was as low as possible.

Disease Control and Survival Outcomes

After a median follow-up of 41.3 months (range, 19.3-71.4 months), a total of 18 patients (78.3%) had developed a locoregional recurrence. For the whole cohort, 1-and 2-year LFFS were 34.1% [95% CI: 18.8%-61.9%] and 18.3%[7.1%-47.1%], respectively (Figure 3A). There was no difference in LFFS between patients who did undergo surgery before reRT and those who did not (Figure 4A). For the IMRT group, 1-and 2-year LFFS was 12.1% [95% CI: 2.0%-75.0%] and 12.1%[2.0%-75.0%], respectively. For the PT group, 1-and 2-year LFFS was 50.0% [95% CI: 28.4%-88.0%] and 22.2%[0.7%-68.9%], respectively (Figure 5A). Among these 18 patients with locoregional failures (LRF), the LRFs occurred within the 95% isodose lines (in-field recurrences) for eight patients (44.4%) and was out-of-field for seven patients (38.8%) (Figure 2). One patient had marginal LRF and for two patients, LRF was detected by clinical examination only (without imaging before death). There were no differences between the two groups for the proportions of "in field" or "marginal" recurrences.

In the whole cohort, over the follow-up period, 17 patients (73.9%) died. For the whole cohort, 1-and 2-year OS was 60.9% [95% CI: 43.9%-84.5%] and 42.5%[26.2%-69.1%], respectively (Figure 3B). There was no difference in OS between patients who did undergo surgery before reRT and those who did not (Figure 4B). For the IMRT group, 1-and 2-year OS was 27.3% [95% CI: 10.4%-71.6%] and 27.3%[10.4%-71.6%], respectively. For the PT group, 1-and 2-year OS was 91.7% [95% CI: 77.3%100%] and 55.6%[32.5%-95.0%], respectively (Figure 5B). For the whole cohort, 1-and 2-year DFS was 30.4% [95% CI: 16.4%-56.5%] and 16.3%[6.2%-24.3%], respectively (Figure 3C). For the IMRT group, 1-and 2-year DFS was 9.1% [95% CI: 1.4%-58.9%] and 9.1%[1.4%-58.9%], respectively. For the PT group, 1-and 2-year DFS was 50% [95% CI: 28.4%-88.1%] and 22.2%[7.2%-68.9%], respectively (Figure 5C). DFS was significantly longer in the PT group (p = 0.031).

In univariate analysis, the location of the 1st relapse (before reRT), histology, surgery before reRT, chemotherapy before reRT, time between the initial RT and reRT, radiation dose, or radiation volume were not significantly associated with locoregional control and overall survival (Supplementary Table 2). LFFS and OS were higher in the PT group, although the difference was not statistically significant (p = 0.16 and p = 0.17, respectively).

Acute toxicities

Dysphagia, dysgeusia, dermatitis, mucositis, and hearing loss were the most frequent acute toxicities observed in our cohort. Five patients (21.7%) experienced grade 3 acute toxicity (dysphagia, dermatitis, and mucositis) (Supplementary Table 3). The comparison between patients in the IMRT and PT groups revealed that severe dysphagia (grade >2) was significantly more frequent in the IMRT group (p = 0.006). Severe dermatitis was also more frequent in the IMRT group than in the PT group, although the difference was not statistically significant (p = 0.093). In contrast, hearing loss was significantly more frequent in the PT group than in IMRT group (p = 0.004). This result could be explained by the slightly different locations of the tumors treated with PT (skull base) and those treated with IMRT (see Section 4).

Late toxicities

Fibrosis, dysphagia, xerostomia and trismus were the most frequent late toxicities observed in our cohort (Supplementary Table 4). Ten patients (43.5%) experienced grade 3 late toxicity. Among the 23 patients, three (13%) experienced carotid dissection, one in the IMRT group and two in the PT group. Two were truly radiation induced carotid blowouts, whereas the third was rather related to progression of a tumor that completely encompassed the vessel. These three patients died of cataclysmic hemorrhage. Besides this, two patients had grade 1 (asymptomatic) temporal radionecrosis and three patients had symptomatic temporal radionecrosis (vertigo, headaches). Two patients had asymptomatic osteoradionecrosis and two patients had symptomatic osteoradionecrosis. Two patients from the PT group developed grade 1 permanent facial paralysis. Three patients, from the PT group, developed grade 1 visual impairment.

Late toxicities in the IMRT and PT groups are not comparable from a statistical point of view because the followup times after reirradiation were different. A representation of these data as Kaplan Meier curves showed no statistically significant difference between the two groups (Supplementary Figure 1).

Discussion

In the present study, we have reported the outcomes and toxicity of a cohort of patients all reirradiated at curative intend for a large local recurrence of HNSCC.

A recent meta-analysis, which included 17 trials and evaluated reRT with IMRT for HNC, showed that LFFS and OS ranged from 36% to 65%, and from 32% to 59%, respectively.23This is consistent to the results obtained in several previously reported studies (Table 3). 13, 15, 20, 24-36 In the whole cohort of our study, 24-month LFFS and OS were 18.3% and 42.5%, respectively. The difference observed in our series for LFFS could be due to the strict selection of patients with only HNSCC and locally advanced disease (73.9% stage III-IV), with a median reRT CTV and PTV of 49.8 and 124.3 cc, respectively. Indeed, Takiar et al. 13 showed that local control was more difficult to achieve with HNSCC than with any other histology. Moreover, in a prospective study including 21 patients reirradiated for a recurrence of HNC, Chen et al. showed that the only identified parameter predictive of local recurrence was the planning target volume.27 Patients with a tumor volume > 27 cm3 had a worse prognosis. Besides this, in the recent MIRI collaborative group's study, surgery, interval between the two courses of irradiation, and organ dysfunction (tumor-related trach or feeding tube dependence) appeared to be prognostic factors for OS.36 From recursive partitioning analysis, the patients with >2 years from the first treatment with resected tumors regardless of final margin status, and without severe dysphagia had the best prognostis. Actually, in the Riaz et al. study,30 surgical resection significantly improved overall survival (p < 0.001 in multivariate analysis) and in the Phan et al. study,25 this interval was an independent factor of recurrence.

Probably due to the small number of patients included in our study, the salvage surgery and the median time between initial irradiation and reRT were not significantly associated with locoregional control and overall survival. Nevertheless, when we applied the MIRI classes in our population (Supplementary Figure 2), the two patients corresponding to RPA MIRI class III had a significantly shorter 2-years OS than the patients of the two other classes (p < 0.001). The 2-years OS of patients in class I and II were not significantly different: 0.5 [95% CI: 18%-100%] and 0.45 [26%-78%]. The number of patients included in our series was probably too small to draw conclusions about the prognostic difference between class I and II (4 patients in class I). Nevertheless, the OS of patients in class II (17/23) was similar to that observed in the MIRI study (0.40 [CI: 33.9%-47.2%]). It is interesting to note that the 11 patients of our cohorts re-irradiated with proton belonged to this MIRI RPA class II. Although not statistically significant, the 2-years OS of patients in this class II re-irradiated with proton was better than that of patients in this class II re-irradiated with photon: 0.53 [95% CI:0.30-0.94] and 0.33 [0.15-0.74], respectively (Supplementary Figure 3).

In our cohort, fibrosis, dysphagia (26.1% grade ≥3), xerostomia and trismus were the most frequent late toxicities, and three patients experienced carotid dissection. Whatever the indication for reRT, acute and especially late toxicities were much more frequent and more severe than after the first irradiation. For instance, in the RTOG 9911 trial reporting the outcomes of 105 patients who received IMRT for a recurrent HNC, the incidence of grade 3/4 late adverse effects was 33.8%, and the incidence of treatment-related deaths was 8%.14 In the Takiar et al. study,13 5-year ≥ grade 3 toxicities were about 50%, and three patients experienced grade 5 toxicity (death). The most common grade 3 toxicities were dysphagia, odynophagia requiring feeding tube placement, osteoradionecrosis, and esophageal stricture. In the meta-analysis of Lee et al.,36 grade ≥3 late toxicities ranged from 14.2% to 57.1%, whereas grade 5 late toxicities ranged from 1.3% to 7.4%. The most common cause of grade 5 complications was carotid rupture, followed by soft tissue necrosis. Furthermore, Romesser et al. 37 have recently shown that PT is a safe alternative to IMRT as it reduces the risk of late toxicity.

In our cohort, dysphagia was more frequent in the IMRT group, whereas hearing loss and temporal radionecrosis were more common in the PT group. This can easily be explained by the location of the reirradiated recurrence: the tumors treated with PT were more frequently in the skull base, whereas the tumors treated with IMRT were often close to the pharyngeal muscles.

To our knowledge, there are currently no dose constraints specific to the context of reirradiation for curative purposes for HNC. In the Takiar et al. study,13 dose constraints are often at the discretion of the clinician (except for the parotid: Mean dose <26 Gy). In our cohort, cumulative doses to OAR often far exceed the recommended dose constraints for initial irradiation. For example, the average dose received by the parotid glands was 64 Gy [range: 23-120] while the dose usually recognized as causing severe xerostomia is 26 Gy.38In a recent study, Embring et al. showed an association between the cumulative dose to (OAR) and the risk of developing osteoradionecrosis and carotid blowout. Their results supported the existing dose constraint for the carotid arteries and bones of 120 Gy.39Furthermore, in our study, the OAR dose constraints for reRT were chosen by assuming an empirical "forgetting factor" of 5% per year for nerve structures.22 This is in agreement with the approach used by Takiar et al. that applied a dose tolerance recovery of 50% for a retreatment interval ≥ 12 months.13 Besides this, it should be noted that in our study, at least for hearing loss, the majority of patients had hearing loss before the start of reRT, and the dosimetric study confirmed that the dose to the inner ear was very low during reRT. From these results, it seems important to take into account the doses received during the first irradiation when planning the re-irradiation.

Moreover, in our study, among the 18 patients with locoregional failures (LRF), the LRFs occurred within the 95% isodose lines (in-field recurrences) for eight patients (44.4%) and was out-of-field for seven patients (38.8%). These results are consistent with the recent pattern-of-failure study of Margalit et al who reported that approximately half of these second local recurrences occurred within the re-irradiated GTV, while the other half occurred outside the field (34%) or at the margin (14%) of the isodose line of prescription.20In this particular context of reRT, the target volumes were chosen to be as small as possible in order to limit the dose to OAR as much as possible. In most cases, CTVs include the GTV/ tumor bed with no margin or with a narrow margin (≤0.5 cm) of normal-appearing surrounding tissue.12 Moreover, multimodal imaging and radiomics could be very useful to help define target volumes with respect to potential sites of recurrence. For instance, Akram et al.40 compared on MRI the recurrent and the non-recurrent regions of 14 NPC patients with histologically proven "in field" recurrence following curative intent IMRT. Seven features were significantly different between recurrent and non-recurrent regions, suggesting that these features may help to better define the target volume before irradiation. Thus, to reduce the risk of second recurrences, especially marginal recurrences, it seems very useful to define this target volume with the help of a radiologist and, in the postoperative setting, with the surgeon.

The main limitations of our series are that it is retrospective, single-center and included a small number of patients. This small number of patients is explained by the rigorous selection procedure: all included patients had to have been treated for an HNSCC with curative intent, and to have a minimum follow-up of 4 months. In addition, more than half of the patients were treated with proton therapy for advanced recurrence of skull base disease. Finally, we studied clinical features, such as disease control and toxicity, and conducted a detailed search for patterns of failure as well as a dosimetric study to understand the toxicity observed.

As a conclusion, curative reRT in HNC is possible for selected cases, but the local recurrence rate in the irradiated field and the risk of toxicity grade ≥2 remain high. Improved selection criteria and more precisely defined target volumes may improve the outcomes of these patients. Proton therapy, essentially for skull base tumors, seems to procure improved locoregional control and overall survival compared to IMRT. These results require confirmation in a prospective randomized study. A B

Figure 1

 1 Figure 1

Table 3 :

 3 Prognostic factors related to locoregional control and overall survival

	Variable	Locoregional control		Overall survival
		n	HR	CI95% (HR)	p value n	HR	CI95% (HR)	p value
	Location of 1 st relapse (before reRT)
	Nasopharynx/skull base	10	1		0.376	10	1		0.414
	Nodes	5	1.56 [0.46 -5.24]		5	0.83	[0.23 -3.01]
	Oropharynx	5	2.11 [0.58 -7.69]		5	0.86	[0.17 -4.32]
	Other	3	0.52 [0.1 -2.64]		3	2.22	[0.66 -7.48]
	Histology
	SCC	12	1		0.418	12	1		0.361
	UCNT	11	0.68 [0.27 -1.74]		11	0.64	[0.24 -1.68]
	Surgery before reRT
	No	14	1		0.284	14	1		0.242
	Yes	9	1.68 [0.64 -4.42]		9	1.78	[0.67 -4.74]
	Chemotherapy before reRT
	No	16	1		0.703	16	1		0.542
	Yes	7	0.82 [0.31 -2.22]		7	0.72	[0.25 -2.08]
	Intervals between initial RT and reRT 23	1	[1 -1.01]	0.86	23	1	[0.99 -1]	0.632
	ReRT technique
	Protons	12	1		0.16	12	1		0.173
	Photons	11	2.24 [0.86 -5.83]		11	1.95	[0.74 -5.16]
	Cumulative dose (1 st RT et reRT)	23	0.94 [0.84 -1.05] 0.256	23	0.95	[0.84 -1.08] 0.451
	Volumes (reRT)
	CTV	23	1.01 [1 -1.01]	0.002	23	1.01	[1 -1.01]	0.006
	PTV	23	1	[1 -1.01]	0.198	23	1	[1 -1]	0.9
	Target volume coverage: V95% CTV 23	0.19 [0.01 -4.21] 0.284	23	0.37	[0.01 -9.55] 0.545
	Target volume coverage: V95% PTV 23	0.44 [0.02 -11.46] 0.618	23	0.22	[0.01 -6.15]	0.366

Pattern of failure study. The locations of the second recurrences (red line) were compared to the 95% isodose of the reirradiated GTV (green line) and classified as "in-field" (A), "marginal" (B), or "outside" (C).


A V Kabashin

S M Klimentov

V Yu Timoshenko

A A Fronya

and International School for Young Scientists on "Physics, Engineering and Technologies for Biomedicine"

International Symposium and International School for Young Scientists on "Physics, Engineering and Technologies for Biomedicine" (PhysBioSymp) is an annually held event in National Research Nuclear University MEPhI (https://eng.mephi.ru/) (Moscow, Russia) since 2016. This symposium is conceived as the main conference of the Institute of Engineering Physics for Biomedicine (PhysBio, https://physbio.mephi.ru/), which is one of new strategic academic units founded in 2016 in the course of transformation of MEPhI from the technical to a global university in order to extend its portfolio toward life sciences, chemical and biomedical engineering. Following the chosen strategy, PhysBio aims at the advancement of its international reputation in biomedical sciences and technologies, as well as the integration of latest research achievements into the educational process in order to contribute to the solution of global problems, such as early diagnostics and efficient therapy of socially significant diseases, including cancer. Conceived as an essentially interdisciplinary institution, PhysBio is unique in combining powerful background of MEPhI in physics, mathematics, engineering, material sciences, nanotechnologies and expertise in new areas in chemistry and biology in order to develop breakthrough technologies for biomedical applications.

The research agenda of PhysBio Institute includes: development of novel materials and techniques for nanomedicine, including nano-theranostics; advanced technologies for nuclear nanomedicine; elaboration of innovative methods for diagnosis and therapy of oncological diseases using novel biocompatible and biodegradable nanomaterials as vectors of targeted delivery of radionuclides; intellectual computer systems of pattern recognition aiming at early cancer diagnosis; technologies of bioprinting and tissue engineering.

Collected in this digest are the materials of the annual PhysBio Symposium and School held in October 26-30 2019 in Moscow which aimed at bringing together leading researchers, high level engineers and experts in biophysics, bio-photonics, nuclear medicine and nano-medicine, in order to present their recent achievements and initiate the exchange of ideas. Within this event, the School included lectures addressed to students and young scientists, whose activities are related to life sciences and medicine. Poster sessions were actively used during the event to give a chance to students and young researchers to present their results. The area of expertise of PhysBioSymp are strongly interlinked to the research agenda of PhysBio and cover the following topics: The program of the PhysBioSymp 2019 conceived 51 plenary lectures and 75 poster talks given by the participants from 10 countries, including Russia, France, Czech Republic, Germany, USA, UK, India, Brazil, China and Kazakhstan.

The meeting was held under supervision of the Russian Ministry of Science and Higher Education, the Russian Ministry of Health, the State Atomic Energy Corporation and was organized in close collaboration with the research institutes of the Russian Academy of Science, National Medical Research Radiology Center, and Kaluga Pharmaceutical Cluster. We acknowledge support from the Russian Science Foundation (RSF, for conducting this international event and publishing the digest. Taking opportunity of publishing in the current issue of IOP Conference Series, we included here few articles presented by young scientist after the poster session of Summer School "Biophotonics" (Moscow, MEPhI, June 20-26, 2020) which has been supported by the same grant #19-72-30012 from RSF. We are very grateful to the invited lecturers, participants, members of the Program and Organizing Committees for their support of PhysBioSimp, and to everyone who contributed to organization of the event, reviewing of the articles and preparation of this digest.

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  Advanced materials and methods for MRI and PET • Bioimaging technologies and materials • Bio-photonics for diagnosis and therapy • Bioprinting • Brachy-, Proton and Ion therapy methods • Diagnosis methods, today and in the future • Immuno-therapy • Isotopes for medical applications • Medical-biological aspects of radiation effects • Nanomaterials for biomedical applications • Plasma and laser technologies for biomedicine


Ahmed Al-Kattan
email: ahmed.al-kattan@univ-amu.fr

Anton A Popov

Plasmonic Si@Au core-satellite nanoparticles prepared by laser-assisted synthesis for photothermal therapy

We describe a laser-assisted methodology for the fabrication of Si@Au core-satellite nanostructures for photothermal therapy applications. The methodology consists in laser ablative synthesis of Si and Au NPs in water/ethanol solutions, followed by a chemical modification of the Si NPs by APTMS and their subsequent decoration by the Au NPs. We show that despite a relatively small size (< 40 nm) the formed core-satellites exhibit a strong plasmonic absorption peak centred around 610 nm and having a large tail over 700 nm overlapping with the first optical window of relative tissue transparency. Being relatively small and exempt of any toxic impurity due to ultraclean laser synthesis, the fabricated nanoparticles promise a major advancement of imaging and phototherapy modalities based on plasmonic properties of nanomatererials.

Introduction

Photothermal hyperthermia is one of exciting implementations of light-induced therapy, which consists in overheating of malignant tissues resulting in its destruction or activation of immune response against tumor. Plasmonic nanomaterials can efficiently sensitize external light stimulus very locally, thus drastically decreasing collateral damage to healthy tissues during a hyperthermic procedure. However, classical plasmonic nanomaterials based on simple spherical Au nanoparticles (NPs) are hardly useful for these tasks, as the plasmonic absorption band of these NPs is centered around 520-550 nm, which is far from the window of relative tissue transparency (650-1000 nm). This plasmonic mismatch problem can partly be solved by engineered Au-based nanoarchitectures such as Au nanorods [START_REF] Huang | Cancer cell imaging and photothermal PhysBioSymp[END_REF] or dielectric-Au core-shell NPs [START_REF] Hirsch | Nanoshell-mediated near-infrared thermal therapy of tumors under magnetic resonance guidance[END_REF][START_REF] Loo | Immunotargeted nanoshells for integrated cancer imaging and therapy[END_REF]. Plasmonic absorption of such complex nanostructures can be tuned toward the transparency window by changing the aspect ratio of the nanorods or the ratio of the shell thickness to core diameter in core-shells. The engineered Au-based nanorods and core-shells have already demonstrated high efficiency as sensitizers of light-induced hyperthermia of cancer tissues, both in vitro and in vivo [START_REF] Huang | Cancer cell imaging and photothermal PhysBioSymp[END_REF][START_REF] Hirsch | Nanoshell-mediated near-infrared thermal therapy of tumors under magnetic resonance guidance[END_REF][START_REF] Loo | Immunotargeted nanoshells for integrated cancer imaging and therapy[END_REF], but the employment of these nanostructures is not free of problems and challenges. The main problems of such nanostructures are their residual toxicity arising due to contamination by by-products during conventional chemical synthesis [START_REF] Hirsch | Nanoshell-mediated near-infrared thermal therapy of tumors under magnetic resonance guidance[END_REF][START_REF] Loo | Immunotargeted nanoshells for integrated cancer imaging and therapy[END_REF][START_REF] Dasgupta | Shape and orientation matter for the cellular uptake of nonspherical particles[END_REF][START_REF] Oldenburg | Nanoengineering of optical resonances[END_REF], elongated shape of the nanorods [START_REF] Dasgupta | Shape and orientation matter for the cellular uptake of nonspherical particles[END_REF]6] and relatively large size of SiO2-Au core-shells [START_REF] Hirsch | Application of INAA to the build-up and clearance of gold nanoshells in clinical studies in mice[END_REF], which can complicate their transport in the organism and excretion from it. The required red-shift of the plasmonic peak at smaller core-shell sizes could be achieved by using a core material with high refractive index such as silicon (Si) instead of silica (SiO2) [START_REF] Lv | Multifunctional core-shell nanoparticle suspensions for efficient absorption[END_REF][START_REF] Chaabani | Comparative theoretical study of the optical properties of silicon/gold, silica/gold core/shell and gold spherical nanoparticles[END_REF]. However, this is typically achieved by complicated chemical methods.

Alternative physical method based on pulsed laser ablation in liquids can offer a simple solution of the problem related to a residual contamination of nanomaterials [START_REF] Kabashin | Nanofabrication with pulsed lasers[END_REF][START_REF] Zhang | Laser synthesis and processing of colloids: fundamentals and applications[END_REF] and complexity of their synthesis. This approach is based on natural formation of smallest clusters under irradiation of a solid target by laser beam. The clusters can then then be deposited on a substrate to form a nanostructured layer [START_REF] Kabashin | Visible Photoluminescence from Nanostructured Si-Based Layers Produced by Air Optical Breakdown on Silicon[END_REF][START_REF] Kabashin | Laser-induced treatment of silicon in air and formation of Si/SiOx photoluminescent nanostructured layers[END_REF] or form a colloidal NPs solution in a liquid medium [START_REF] Fojtik | Laser Ablation of Films and Suspended Particles in Solvent-Formation of Cluster and Colloid Solu-tions[END_REF][START_REF] Dolgaev | Nanoparticles Produced by Laser Ablation of Solids in Liquid Environment[END_REF][START_REF] Kabashin | Synthesis of Colloidal Nanoparticles during Femtosecond Laser Ablation of Gold in Water[END_REF]. This method does not require any chemicals and can be performed in an ultrapure liquid such deionized water excluding any residual contamination of the nanoparticle surface, which is critically important for projected biomedical applications [START_REF] Kabashin | What Theranostic applications could ultrapure lasersynthesized Si nanoparticles have in cancer?[END_REF][START_REF] Kabashin | Laser-processed nanosilicon: a multifunctional nanomaterial for energy and healthcare[END_REF]. As an example, our group recently elaborated the technique of femtosecond (fs) laser ablation in water and organic media for controllable synthesis of NPs from a variety of materials, including Au [START_REF] Maximova | Size-Controllable Synthesis of Bare Gold Nanoparticles by Femtosecond Laser Fragmentation in[END_REF][START_REF] Bailly | In vivo evaluation of safety, biodistribution and pharmacokinetics of laser-synthesized gold nanoparticles[END_REF], Si [START_REF] Baati | Ultrapure laser-synthesized Si-based nanomaterials for biomedical applications: In vivo assessment of safety and biodistribution[END_REF][START_REF] Al-Kattan | Ultrapure Laser-Synthesized Si Nanoparticles with Variable Oxidation States for Biomedical Applications[END_REF][START_REF] Petriev | Nuclear nanomedicine using Si PhysBioSymp[END_REF], TiN [START_REF] Petriev | Nuclear nanomedicine using Si PhysBioSymp[END_REF][START_REF] Popov | Laser-synthesized TiN nanoparticles as promising plasmonic alternative for biomedical applications[END_REF] NPs. Laser ablation can also be used for the synthesis of complex structures such as core-shells, non-equilibrium alloys and mixed phase NPs. As an example, we recently successfully demonstrated fs laser ablative synthesis of contamination-free Si-Au core-shell nanostructures [START_REF] Zelepukin | Laser-synthesized TiN nanoparticles for biomedical applications: evaluation of safety, biodistribution and pharmacokinetics[END_REF]. We found that such nanostructures, despite having a relatively small size (less than 50 nm), have plasmonic absorption feature in the window of relative tissue transparency.

In this paper we provide details about optimization of the synthesis protocol of such Si-Au core-shell NPs to achieve maximum red shift of the plasmonic feature. We believe that this nanostructures with exceptional purity could provide a major advancement in biomedical applications.

Experimental methods

Solutions of colloidal Si NPs were obtained from 0.5 µm Si micropowder, which was preliminary prepared by mechanical milling of a Si wafer as it was described in [START_REF] Baati | Ultrapure laser-synthesized Si-based nanomaterials for biomedical applications: In vivo assessment of safety and biodistribution[END_REF][START_REF] Al-Kattan | Ultrapure Laser-Synthesized Si Nanoparticles with Variable Oxidation States for Biomedical Applications[END_REF]. Si powder was dispersed in liquid by a ultrasonication for 15 minutes in a 4.5 mL vial at 0.4 mg/mL concentration. Three liquids were used: pure anhydrous ethanol (100% ethanol), 1:1 water/ethanol mixture (50% ethanol) and pure water (0% ethanol). Si micropowder solutions were then fragmented for 60 minutes using radiation from a Yb:KGW femtosecond laser (Amplitude systems, France, wavelength 1025 nm, repetition rate 10 kHz, pulse dura-tion 480 fs). The laser beam was focused in the center of a vial filled by micropowder solutions using a 75 mm lens. The homogenization of the solution was done by a magnetic stirrer. Unfragmented Si powder was removed by the centrifugation at 3500 rpm/min for 20 minutes leaving Si NPs at ~0.1 mg/mL concentration. Au NPs were synthesized using the same laser in the ablation geometry, as previously described in [START_REF] Maximova | Size-Controllable Synthesis of Bare Gold Nanoparticles by Femtosecond Laser Fragmentation in[END_REF][START_REF] Bailly | In vivo evaluation of safety, biodistribution and pharmacokinetics of laser-synthesized gold nanoparticles[END_REF][START_REF] Sylvestre | Surface Chemistry of Gold Nanoparticles Produced by Laser Ablation in Aqueous Media[END_REF]. For this, a gold target (99.99%, Sigma Aldrich) immersed in 7 mL of 0.1mM aqueous NaCl solution was irradiated by a laser beam focused by the same 75 m lens. To ensure a homogenous ablation process, the ablation target was continuously moved with 2 mm/s speed using motorized linear translational stages (Newport).

The functionalization step was performed by adding a solution of 3-aminopropyltrimethoxysilane (APTMS) (Sigma Aldrich) to Si NPs solutions at ambient condition. The obtained mixture was stirred at room temperature for 24 hours. Then aqueous solutions of Au NPs were gently added to Si-APTMS NPs solutions and additionally stirred at ambient conditions for 24 hours.

A transmission electron microscopy (TEM) system (JEOL JEM 3010) operating at 300 kV was employed to perform structural characterizations (size distribution, crystalline structure, morphology) of Si-Au nanostructures. Samples for the electron imaging were prepared by placing a drop of a NPs solution on a carbon coated copper TEM grid (200 mesh, Oxford instruments) and its drying at ambient conditions. Statistical data were obtained based on analysis of size characteristics from several hundreds of NPs. ζ-potential measurements were performed using a Zetasizer Nano ZS system (Malvern Instruments). Optical extinction spectra of the NPs were measured by a UV-Vis spectrophotometer (UV-2600, Shimadzu) using 10 mm optical path length quartz cuvettes.

Results and discussion

Laser ablation of a Si target resulted in crystalline Si NPs having spherical shape with lognormal size distribution and mode size of about 30 ± 3 nm as follows from TEM and SAED analysis (data not shown). At the same time, Au NPs prepared by laser ablation in 0.1 mM aqueous NaCl solutions also had a spherical shape with normal size distribution and mean size 4 ± 0.8 nm. As follows from the electron diffraction pattern (data not shown), the formed Au NPs had FCC lattice.

To overcome electrostatic repulsion of Si and Au NPs, which are both negatively charged (-45 ± 1.5 mV and -32 ± 2.6 mV, respectively), and to form core-shell structures we functionalized surface of Si NPs by an organosilane molecule 3-aminopropyltiethoxysilane (APTMS), which is widely used to coat organic NPs and functionalize them with a variety of molecules. APTMS is a linear chain molecule with two alkoxy functional groups on one end and an amine functional group on the other end. Hydrolysis of methoxy groups of APTMS followed by their reaction with the hydroxyl Si-OH group on the oxidized Si NPs surface results in formation of Si-O-Si linkages and firm binding of APTMS molecules at the Si NPs surface. At neutral or acidic conditions amine group of the APTMS molecules is protonated, which renders positive surface charge of functionalized Si NPs. This was confirmed experimentally by measuring Zeta-potential. The potential of Si NPs increased from -45 ± 1.5 mv to 2.58 ± 0.20 mV after functionalization by APTMS. Therefore, the functionalization makes possible the electrostatic coupling between positively charged functionalized Si NPs and negatively charged Au NPs [START_REF] Westcott | Formation and adsorption of clusters of gold nanoparticles onto func-tionalized silica nanoparticles[END_REF].

The formed nanostructures had core-satellite geometry with a large Si core decorated by small Au NPs as shown in Figure 1. The Si cores of Si-Au core-shells were fully coated with a continuous shell of randomly distributed Au NPs, while separated Au NPs were rare in the solution. Statistical analysis of the size of nanostructures showed that the mean size of silicon cores was about 37 nm, while the thickness of Au NPs-based shell was about 4 nm, with the mean size of core-shells close to 40 nm, which matched the original size of Si and Au NPs. To improve grafting and reduce self-polymerization of APTMS molecules, we optimized physicochemical parameters of the coupling process. We found that 80 µL of APTMS per 4.5 mL of fresh colloidal solution of Si NPs (concentration 0.1 mg/mL) provides the most stable NPs solutions without signs of precipitation, while excess of APTMS results in jellification. Then we found that successful formation of Si-Au core-shells requires significant excess of Au NPs. This was found by mixing functionalized Si NPs and Au NPs in concentrations 0.1 mg/mL in different ratios, namely 1:1, 1:10, 1:30 (Si:Au). The 1:30 ratio provided the largest red shift of the plasmonic feature.

We finally studied dependence of plasmonic peak position on the liquid medium in which APTMS molecules were grafted to Si NPs surface. For this we separately functionalized Si NPs in pure anhydrous ethanol (100% ethanol), 1:1 water:ethanol mixture (50% ethanol) and in pure water (0% ethanol). The results are shown in Figure 2. As one can see, the increase of ethanol content resulted in increase of red shift of the plasmonic peak. In particular, at 0% ethanol (pure water) position of the plasmonic peak of the core-shell structures was almost identical to the position of the plasmonic peak of pure Au NPs. This mean that core-shell NPs did not form under such conditions most probably due to fast hydrolysis of methoxy functional groups of APTMS molecules and their self-polymerization before reaching surface of Si NPs. At the same time, Si-Au core-shell NPs prepared in 100% (anhydrous) ethanol had the largest red shift of the plasmonic peak, which was located close to 610 nm.

SiNPs-APTMS-AuNPs in 100% Ethanol SiNPs-APTMS-AuNPs in 50% Ethanol SiNPs-APTMS-AuNPs in 0% Ethanol With ratio SiNPs:AuNPs 1:30 Figure 2. Extinction spectra of Si-Au core-satellite nanocomposites conjugated in different solvents: 100 % ethanol (black curve, anhydrous ethanol), 50% ethanol (red curve, 1:1 water: ethanol mixture) and 0% ethanol (blue curve, pure water).

Thus, by applying a chemical protocol on laser synthesized Si and Au nanomaterials, we managed to assemble core-satellite Si@Au nanostructures exhibiting a much red-shifted plasmonic extinction peak compared to spherical Au nanoparticles (610 nm compared to 520-550 nm). We suppose that the formed core-satellite structures are promising objects for photothermal therapy of cancer. As one of main advantages for such applications, we see exceptional purity of formed nanoformulations due to a complete avoidance of the chemical reduction step. Indeed, Si and Au NPs are produced separately by femtosecond laser ablation in deionized water, which leads to the formation of bare (ligand-free) surface in the absence of any contamination by toxic products. Laser-synthesized Au and Si NPs present highly safe objects for biological systems, as we showed in previous tests in vitro and in vivo [START_REF] Bailly | In vivo evaluation of safety, biodistribution and pharmacokinetics of laser-synthesized gold nanoparticles[END_REF][START_REF] Baati | Ultrapure laser-synthesized Si-based nanomaterials for biomedical applications: In vivo assessment of safety and biodistribution[END_REF][START_REF] Al-Kattan | Ultrapure Laser-Synthesized Si Nanoparticles with Variable Oxidation States for Biomedical Applications[END_REF][START_REF] Petriev | Nuclear nanomedicine using Si PhysBioSymp[END_REF]. Furthermore, laser-synthesized Si NPs are biodegradable as in biological environment they decay and excrete from the body with the urine within several days [START_REF] Al-Kattan | Ultrapure Laser-Synthesized Si Nanoparticles with Variable Oxidation States for Biomedical Applications[END_REF]. A subsequent application of chemical protocol on the basis on APTMS can not add any toxicity as APTMS is a highly biocompatible product demonstrated in various biomedical applications [START_REF] Westcott | Formation and adsorption of clusters of gold nanoparticles onto func-tionalized silica nanoparticles[END_REF]. Another expectation is related to possible gradual decay of core-satellites under long-term exposition to biological environment and a complete excretion of decay products from the organism.

It should be noted that laser-ablation approached has already been applied for the synthesis of Aubased core-shells, using spontaneous formation of core-shells during the ablation or co-ablation of materials having appropriate physico-chemical properties [START_REF] Amendola | Magneto-plasmonic Au-Fe alloy nanoparticles designed for multimodal SERS-MRI-CT imaging[END_REF][START_REF] Lin | Laser synthesis of gold/oxide nanocomposites[END_REF][START_REF] Jo | Formation of core-shell micro/nano particles through pulsed-laser deposition in liquid[END_REF], or by the combination of laser ablation with an additional chemical reduction step using HAuCl4 solutions [START_REF] Jiménez | Laser-ablationinduced synthesis of SiO2-capped noble metal nanoparticles in a single step[END_REF][START_REF] Liu | Fabrication of Si/Au core/shell nanoplasmonic structures with ultrasensitive surface-enhanced Raman scattering for monolayer molecule detection[END_REF]. However, the first laser-ablative approach typically leads to a wide size and shape dispersion of formed composites, while the second combination approach still cannot avoid residual contamination by side products of the chemical reduction process. In this sense, the combined methodology proposed in our study is capable of avoiding problems of both approaches.

We believe that the core-satellite geometry of Si-Au nanocomposites can be advantageous for these applications compared to classical SiO2-Au core-shell nanostructures [START_REF] Hirsch | Nanoshell-mediated near-infrared thermal therapy of tumors under magnetic resonance guidance[END_REF][START_REF] Loo | Immunotargeted nanoshells for integrated cancer imaging and therapy[END_REF][START_REF] Oldenburg | Nanoengineering of optical resonances[END_REF] due to a much smaller size. Indeed, the diameter of core-satellites is less than 40 nm, which is in the range of relative safety of nanomaterials for biological systems and should favor transport of nanostructures in vivo to reach affected organs and their easier removal from the organism. Besides, the presence of laser-ablated Si nanostructures in the composition of core-satellites can enable a series of novel theranostic functionalities, including the generation of non-linear response for bioimaging [START_REF] Kharin | Bi-modal nonlinear optical contrast from Si nanoparticles for cancer theranostics[END_REF], hyperthermia-based therapies under radiofrequency [START_REF] Tamarov | Radio frequency radiation-induced hyperthermia using Si nanoparticlebased sensitizers for mild cancer therapy[END_REF] and photon [START_REF] Oleshchenko | Localized infrared radiation-induced hyperthermia sensitized by laser-ablated silicon nanoparticles for phototherapy applications[END_REF] excitation. As another potential application, we see the use of core-satellites as probes for Surface Enhanced Raman Scattering (SERS) for the identification of trace amounts of biological species. Inverted geometry of Au-Si core-shells, composed of Au core covered by Si shell, has already been reported in our previous study [START_REF] Kögler | Bare laser-synthesized Au-based nanoparticles as nondisturbing surface-enhanced Raman scattering probes for bacteria identification[END_REF]. Exhibiting local electric field enhancement due to the excitation of plasmons around 520 nm, such core-shells demonstrated a high efficiency in the identification of bacteria cultures [START_REF] Kögler | Bare laser-synthesized Au-based nanoparticles as nondisturbing surface-enhanced Raman scattering probes for bacteria identification[END_REF]. We expect that Si-Au core-satellites can provide similar enhancement and be used with red-shifted pumping wavelength (e.g., 630, 785 nm) for SERS excitation, which is preferable under some conditions. It should be finally noted that silicon/gold architecture also presents a very promising platform for surface plasmon resonance biosensing [START_REF] Patskovsky | Near-infrared surface plasmon resonance sensing on a silicon platform[END_REF][START_REF] Patskovsky | Si-based surface plasmon resonance sensing with two surface plasmon polariton modes[END_REF], which can have advantages over classical glass/gold platform [START_REF] Liedberg | Surface plasmon resonance for gas detection and PhysBioSymp[END_REF][START_REF] Kabashin | Surface plasmon resonance bio-and chemical sensors with phase-polarisation contrast[END_REF][START_REF] Law | Wide dynamic range phase-sensitive surface plasmon resonance biosensor based on measuring the modulation harmonics Biosens[END_REF] due to essentially different conditions of plasmon coupling. We believe that the proposed core-satellites can be also explored as nanoscale implementations of such biosensors.

Conclusions

In summary, we described a laser-assisted procedure for the fabrication of Si@Au core-satellite nanostructures, comprising of a Si core covered with small Au nanoparticles (NPs). The methodology employed laser ablative synthesis of Si and Au NPs in water/ethanol solutions, followed by their chemical modification and linking via organsilane APTMS molecules. The core-satellites had a strong plas-monic peak centered around 600-610 nm with a long tail over 700 nm, which overlaps with biological transparency window. Being relatively small and exempt of any toxic impurity due to ultraclean laser synthesis, the fabricated Si@Au core-satellites look as a promising object for cancer theranostics.

Figure 1 .

 1 Figure 1. Statistical analysis of sizes of Si-Au core-shell nanostructures. Insert demonstrates typical TEM image of the core-shell NPs prepared by fs laser ablation in water.

Acknowledgements A.A.-K. thanks the International Associated Laboratory (LIA) MINOS project and GRAVITY project of the ITMO "Plan Cancer 2014-2019" INSERM program. The authors acknowledge contribution of the Russian Science Foundation (Project 19-72-30012) for the elaboration of protocols for the fabrication of highly calibrated nanoparticles.

Conflict of Interests

The authors declare no conflict of interest.


1

Wuman(s) and non-Wuman(s) in medieval China Classifying people in Fan Chuo's Manshu (Book of the barbarians)

Alexis Lycas

Harvard-Yale Symposium Series on Middle Period China, 3 June 2022 (online)

Origins of the project + Outline

Criteria -language

• Endonyms/autonyms: 娑羅籠, 阿等路, 阿旁, 阿竿路, 磨彌殿 + all Nanzhao rulers names 皮羅閣, 閣邏鳳, etc.

• Exonyms: 西蠻, 順蠻, 生蠻, 暴蠻, 兩林, etc.

Criteria -political practices

• 男則髮髻,女則散髮,見人無禮節拜跪,三譯四譯,乃與華通。大部 落則有鬼主,百家二百家小部落,亦有小鬼主。一切信使鬼巫,用相 服制。土多牛馬,無布帛,男女悉披牛羊皮。 (juan 1) • The men tie their hair in a bun, the women leave it loose; [when they] meet other men they do not [observe] the rites and do not bow down; communication with the Chinese is possible only after a triple or quadruple translation. Large tribes have a great master of spirits. Small tribes of one hundred families or two hundred families also have a small spirit master. All their trusted envoys are shaman magicians who use [shamanism] to subdue and control. [For working] the land there are many oxen and horses, they have no clothes and silks, men and women all spread cow or sheep skins on the shoulders.

• 悉皆野蠻,無君長也。地有瘴毒 (juan 2)

• All are wild Man, without a chief, and the land is poisoned with miasma.

•

  Transmission:Yongle dadian 永樂大典 > Siku quanshu 四庫全書 > Xiang Da 向達 (1900-1966) • Translations: Gordon Luce (1961), Walter Stanish (2015-), Bu Shaoxian 卜绍先 (2018) Structure Manshu Song local gazetteer 雲南界內途程第一 Journeys within Yunnan 山川江源第二 Mountains and plateaux, rivers and sources 六詔第三 Six kingdoms-zhao 名類第四 ordering names 六𧸘第五Six prefectures-jian 雲南城鎮第六 Cities and garrisons of Yunnan 雲 南 管 內 物 產 第 七 Products under the administrative purview of Yunnan 蠻夷風俗第八 Customs of the Man barbarians 南蠻條教第九 Laws and statutes of the Southern Man 南 蠻 疆 界 接 連 諸 番 夷 國 名 第 十 Names of the barbarian states bordering the Southern Man 沿革 changes in administrative units 四至 distances to surrounding capitals 疆域 borders 城郭 walls of the administrative seat 鄉村 cantons and villages 山川 mountains and rivers 戶口 population 坊市 urban quarters 橋梁 bridges 津渡 fords 堰閘 water conservancy 鋪驛 postal stations 土產 local products 學校 schools 風俗 customs Criteria -customs • 烏蠻婦人以黑繒為衣,其長曳地;白蠻婦人以白繒為衣,下不過膝。 (juan 1) • The wives of the Black Man make their clothes with black silk, the length of which hangs down to the ground. The wives of the White Man make their clothes with white silk, the bottom of which does not exceed the knee • 尋傳蠻,閣羅鳳所討定也。俗無絲綿布帛,披娑羅籠。跣足可以踐履 榛棘。 (juan 4) • The Xunchuan Man are those who were punished and pacified by Geluofeng. They do not use silk waste to make clothes, they wear [cotton] sarongs. Although they are barefoot, they can trample scrubs.