PhD Nadia Arbouche
email: nadia.arbouche@hotmail.it

MD Alexis Walch

| Jean-Sébastien

Raul Phd

PhD | Pascal Kintz

Intentional overdose of glargine insulin: Determination of the parent compound in postmortem blood by LC-HRMS

Keywords: LC-HRMS, postmortem blood, suicide Highlights

Insulin glargine is a long-acting insulin analog that is converted after enzymatic cleavage of the arginine pair of the β-chain into its main metabolite M1 (21 A -Gly-insulin), which is responsible for the hypoglycemic activity. In all the overdose cases described in the literature, only M1 concentrations have been reported, whereas insulin glargine was always absent or below the limit of quantitation. In this study, we present a case of suicide of a young nurse by injection of insulin glargine in which the parent molecule was found at a toxic concentration in blood. The determination and the discrimination of insulin glargine from human insulin and other synthetic analogs in the blood specimen were performed by liquid chromatography coupled to high-resolution mass spectrometry (Waters XEVO G2-XS QToF) and extraction after precipitation in the presence of bovine insulin (internal standard), with a mixture of acetonitrile/methanol +1% formic acid followed by purification on solid phase extraction cartridges C18.

Glargine insulin tested highly positive in the blood with a concentration of 1.06 mg/L. Due to the difficulty in obtaining a M1 pure standard, the metabolite could not be dosed. This unique presence of the parent molecule, reported for the first time, can be explained by inter-individual variability in the rate of conversion to metabolite.

Intravenous injection versus subcutaneous injection can also explain the presence of insulin glargine. Finally, the dose injected may have been so high that saturation of the proteolytic enzymes responsible for conversion to M1 should have occurred.

| INTRODUC TI ON

Until the beginning of the 20th century, the only possible treatment to fight diabetes consisted of drastic food control while type I diabetic subjects died from this disease. It was not until 1921 that a group of researchers at the University of Toronto succeeded in extracting insulin from the animal pancreas for the first time and studied its action in humans. Since that time, initially insulins of animal origin and later synthetic insulins obtained from genetically modified strains of Escherichia coli have been the most widely used treatment for type I and type II diabetes.

Several formulations of synthetic analogs of human insulin have been proposed according to the expected pharmacokinetic profile.

They can be divided into two categories: fast-acting insulins (lispro, aspart, and glulisine), developed to mimic the rapid and intense pancreatic secretion of insulin after a meal, and long-acting insulins (glargine, detemir, and degludec), characterized by a constant active concentration in the blood over long term with no concentration peak [1].

Insulin glargine (Lantus®) is a long-acting synthetic human insulin used for basal insulin therapy and available for subcutaneous injection as a pre-filled pen in dosages of 100 and 300 units/mL, once a day. It is only intended to be administered subcutaneously, as this is precisely the route of injection that allows its prolonged effect.

The addition of two arginines at the C-terminal position of the β-chain and the replacement of asparagine by glycine at position 21 of the α-chain shifts the isoelectric point of insulin glargine, making it poorly soluble at neutral pH but fully soluble at the acidic pH of the injection solution (pH 4). After injection into the subcutaneous tissue (pH 7.4), the acid solution is neutralized, resulting in the formation of micro-precipitates that represent a "depot" from which small quantities of insulin glargine are continuously released. The addition of zinc to the preparation further stabilizes the product by delaying the release of insulin glargine monomers and dimers, extending its duration of action. As a result, the concentration/time curve is regular, with no peak, and the duration of its action is prolonged [START_REF]Lantus, INN-insulin glargine[END_REF].

After subcutaneous injection of Lantus®, insulin glargine is rapidly degraded at the carboxyl end of the β-chain into two active metabolites, M1 (21 A -Gly-insulin) and M2 (21 A -Gly-des-30 B -Thrinsulin). In plasma, the major circulating metabolite is the M1 metabolite and its concentration increases with the administered dose of Lantus®. The effects of subcutaneous injection of Lantus® are mainly due to exposure to the M1 metabolite, as insulin glargine and the M2 metabolite are almost never detectable after subcutaneous injection of insulin glargine [START_REF] Bolli | Plasma exposure to insulin glargine and its metabolites M1 and M2 after subcutaneous injection of therapeutic and supratherapeutic doses of glargine in subjects with type 1 diabetes[END_REF].

Insulin overdoses are under-reported in the literature despite the large number of diabetics using this therapy. Indeed, diabetes is one of the most common chronic diseases in the world and in 2020, about 3.5 million people in France were treated with antidiabetic drugs, which represents 5.3% of the population [START_REF]Le diabète en France: les chiffres 2020[END_REF].

As the prevalence of diabetes increases, cases of insulin misuse and overdose are also increasing. Compared with the cases described in the 20th century where less than 5% of cases were suicide cases [START_REF] Arem | Insulin overdose in eight patients: insulin pharmacokinetics and review of the literature[END_REF], in the last 20 years, 90% of insulin glargine overdoses were suicide attempts (Table 1).

It is not surprising that insulin is used for suicidal purposes since diabetic patients have a much higher risk of developing depression than the rest of the population [START_REF] Mork | Massive insulin overdose managed by monitoring daily insulin levels[END_REF].

Although at therapeutic doses insulin glargine pharmacokinetics is fairly well described, there is a lack of information in overdose situations due to the limited data on the literature.

It is because of its long-acting properties that hypoglycemia caused by insulin glargine overdose is more problematic than with other synthetic analogs of human insulin, as even if treated early, it may require a long period of follow-up in which moments of normoglycemia and moments of severe hypoglycemia will alternate [START_REF] Ashawesh | Intentional overdose with insulin glargine[END_REF][START_REF] Lu | Lantus insulin overdose: a case report[END_REF].

The causes of prolonged duration of hypoglycemia following glargine overdose could be due to delayed dose-dependent absorption due to the "depot" effect at the injection site and downregulation of insulin receptors [START_REF] Groth | Octreotide for the treatment of hypoglycemia after insulin glargine overdose[END_REF]. Studies have shown that after a therapeutic injection of 0.3 units/kg of insulin glargine, hypoglycemia persists for 24 h, whereas following an injection of 0.4 units/kg, hypoglycemia persists for approximately 30 h [START_REF] Groth | Octreotide for the treatment of hypoglycemia after insulin glargine overdose[END_REF].

These results therefore suggest that the duration of hypoglycemia is probably dependent on the dose of injected insulin glargine.

It can be anticipated that at higher doses, hypoglycemia can last for a longer period.

In the cases described in the literature over the last 20 years, insulin glargine was seldom identified and quantified (Table 1). When identification and dosage were reported, immunoassay methods were generally used. The identification of the human insulin analog involved in the overdose is of critical importance. First, from a clinical point of view, as previously mentioned, insulin glargine overdose can induce recurrent hypoglycemic episodes over a long period of time, and knowing the type of insulin is of great benefit to the clinician in predicting the type and duration of treatment. Second, from a medico-legal point of view in the absence of anamnesticcircumstantial data (medical prescriptions, diabetes, or diabetic family members), the accurate identification can be the only evidence of exogenous insulin administration at the scene. In a 2016 study performed by Kim et al. [START_REF] Kim | Insulin glargine and insulin aspart overdose with pharmacokinetic analysis[END_REF] involving insulin glargine overdose, the metabolite M1 was identified and quantified throughout the hypoglycemic state using a LC-MS/MS technology. However, the authors did not detect the parent molecule. In order to increase the specificity and sensitivity of the method and to avoid any interference with other analogs, it is suitable to use separative methods coupled to mass spectrometry or high-resolution mass spectrometry (HRMS) and to avoid immunoassay tests. To date, HRMS has only been used in a study performed by Yong-Xi Li et al. [START_REF] Li | Quantitation of insulin analogue glargine and its two metabolites M1 and M2 on triple quad 6500 and triple TOF 5600 LC-MS/MS systems in a dog toxicokinetics study[END_REF] on the toxicokinetic of insulin glargine in dogs and was never applied to human biological specimens.

In this report, the authors present a case of suicide by insulin glargine overdose with the first identification of the parent molecule using a high-resolution mass spectrometry system (UHPLC/Q -TOF-MS).

| C A S E REP ORT

A 26-year-old woman (nurse by profession) was found dead at home lying on a sofa. The woman was found in her home by a friend after he received a letter mentioning her suicidal intention.

Several syringes corresponding to insulin injection devices were found next to the body, as well as loxapine capsules. According to what has been reported to the investigators, it seems that the young woman had attempted suicide twice before this fatal outcome.

The body examination and the postmortem body scanner revealed a marked asphyxiation syndrome and nail cyanosis. A tablet corresponding to a blood glucose sensor located on the right arm suggested that the young woman was diabetic. A venous catheter fixed on the left wrist of the corpse was also found. As this was an external examination of the body and no autopsy was performed, no histological data are available. In addition, no biochemical tests were performed, in particular blood glucose, lactates, and ketone bodies, as this type of analysis was not available given the long delay between autopsy and the request of the Prosecutor. However, by directly investigating the toxic molecule involved, indirect measurements of antemortem blood glucose are not necessary.

As the forensic pathologist was focused on a toxic death, samples of femoral blood, urine, and hair were collected for toxicological tests.

Blood and urine were stored at +4°C until analysis, while head hair was stored in an envelope at room temperature.

| ME THODS

For the identification and quantification of insulins in blood, a previously validated and published method was used [START_REF] Arbouche | Attempted murder of a young child followed by an attempted suicide of the mother by injection of insulin aspart: identification of quantification of insulin by LC-HRMS and UPLC-MS/MS in blood of the two cases[END_REF]. All the chemical reagents, the extraction procedure and LC-HRMS parameters are reported in the previous published paper.

Since the focus, in this case, was on insulin glargine, we point out that for glargine insulin, the sevenfold charged ion was observed at m/z 867.46288 (which deconvolutes to 6072.24016) and that glargine insulin was eluted at 2.69 min.

TA B L E 1

Case reports of insulin glargine overdose. The methods used to perform the toxicological screening have also been described in the same article [START_REF] Arbouche | Attempted murder of a young child followed by an attempted suicide of the mother by injection of insulin aspart: identification of quantification of insulin by LC-HRMS and UPLC-MS/MS in blood of the two cases[END_REF].

| RE SULTS AND D ISCUSS I ON

The method used for the analysis of the blood sample has been previously validated and published [START_REF] Arbouche | Attempted murder of a young child followed by an attempted suicide of the mother by injection of insulin aspart: identification of quantification of insulin by LC-HRMS and UPLC-MS/MS in blood of the two cases[END_REF].

Briefly, the calibration curves showed a good linearity in the range 0-100 ng/mL with a correlation coefficient ranging from 0.9994 to 0.9999. The LOD and the LOQ are 0.5 and 1 ng/mL, respectively.

Insulin glargine was found in the femoral blood at the concentration of 1.06 mg/L. Figure 1 shows the chromatogram after the extraction of the blood specimen.

Further analyses, achieved during a complete toxicological screening, revealed the presence of tiapride (24.95 mg/L), loxapine (0.18 mg/L), and nordiazepam (0.16 mg/L). While tiapride was at a toxic level, loxapine, and diazepam were considered at therapeutic levels.

Until 20 years ago, the most widely identified antidiabetics after poisoning were biguanides and sulfonylureas. However, in the 2019 report of the American Association of Poison Control Centers, insulin cases have been multiplied by 3, surpassing now sulfonylureas intoxications [START_REF] Watson | annual report of the American Association of Poison Control Centers Toxic Exposure Surveillance System[END_REF][START_REF] Gummin | annual report of the American Association of Poison Control Centers' National Poison Data System (NPDS): 37th annual report[END_REF].

Insulin overdoses, whether accidental or intentional, are potentially fatal due to the alteration of central nervous system (CNS) metabolism induced by hypoglycemia, which can result in cerebral dysfunction, pulmonary edema and if not treated in time and prolonged can lead to coma and neurological death [START_REF] Fromont | Prolonged glucose requirements after intentional glargine and aspart overdose[END_REF]. Our results indicate that the hypoglycemic agent most present in the circulation was insulin glargine.

The presence of other drugs (tiapride, loxapine, and nordiazepam) may have contributed or aggravated the hypoglycemia caused by insulin glargine. In fact, the use of antipsychotics in diabetic patients has been shown to be associated with a higher risk of hypoglycemia [START_REF] Takahiko | Severe hypoglycemia associated with tiapride in an elderly patient with diabetes and psychosis[END_REF].

Insulin overdoses remain a rare occurrence compared with the large number of diabetics under insulin therapy. In fact, the problem may be underestimated and cases under-reported, with hypoglycemia often being mistaken for an accident. This is due both to the inability/difficulty of laboratories to perform the analysis of such derivatives and to the fact that in the absence of anamnesticcircumstantial evidence (being diabetic, having a diabetic relative) the analysis is not even requested by police, judges, prosecutors, and forensic pathologists (Figure 2).

In the literature, the cases of insulin glargine overdose are limited to case reports in which the drug is often not quantified (Table 1).

However, most of the cases described show that the final outcome of an insulin glargine overdose does not depend primarily on the dose injected. According to a literature review by Arem et al. [START_REF] Arem | Insulin overdose in eight patients: insulin pharmacokinetics and review of the literature[END_REF], death can occur even at doses as low as 400 units, while patients at doses up to seven times higher can fully recover [START_REF] Karatas | The highest (3600 IU) reported overdose of insulin glargine ever and management[END_REF][START_REF] Kumar | Management of intentional overdose of insulin glargine[END_REF]. Karatas et al. [START_REF] Karatas | The highest (3600 IU) reported overdose of insulin glargine ever and management[END_REF] described a case of a diabetic patient who, after attempting suicide by injecting himself 3600 units of insulin glargine, required a glucose infusion for about 120 h due to frequent hypoglycemic episodes but recovered without permanent complications. There are several factors that appear to influence the reaction and final outcome of an insulin glargine overdose; the number of the injection sites, diabetes, kidney, and liver dysfunction could reduce gluconeogenesis and the ability to convert insulin and the delay between administration and treatment [START_REF] Fuller | Lantus overdose: case presentation and management options[END_REF][START_REF] Sandooja | Critical low catastrophe: a case report of treatment-refractory hypoglycemia following overdose of long-acting insulin[END_REF]. described in the literature, insulin intake was self-reported and was not documented by subsequent analyses.

Knowledge of the type of insulin used is the key to determining the duration of treatment and the observation period required for the patient's follow-up. In most of the reported cases, insulin characterization was not performed. The latter would be essential to show the rate of decline in insulinemia and to predict when the effects of the overdose will end [START_REF] Mork | Massive insulin overdose managed by monitoring daily insulin levels[END_REF].

A patient may present to the emergency room normoglycemic and develop a hypoglycemic syndrome a few hours later due to a release of insulin from a depot. Identifying the insulin involved at an early stage and monitoring insulin levels is therefore very useful for the treatment but generally, patient management in a clinical context is mainly based on the clinical assessment of signs and symptoms.

While in clinical practice the problem is to identify the insulin analog to start the most suitable treatment, in forensic medicine, it is essential to identify the agent that caused the poisoning without any doubt, given all the legal consequences.

In forensic medicine, the cause of death can be based on circumstantial evidence such as the presence of empty syringes, testimonies, suicide letters, or even insulin/peptide C ratio by means of immunoassays (when requested).

However, immunoassays are not appropriate for postmortem specimens due to the lack of specificity of the antibodies which often fail to distinguish human insulin from its analogs, metabolites, and insulins of animal origin. As a consequence, in the legal context, immunoassays for insulin do not meet the criteria of quality, especially in the absence of complementary anamnestic information [START_REF] Arbouche | Attempted murder of a young child followed by an attempted suicide of the mother by injection of insulin aspart: identification of quantification of insulin by LC-HRMS and UPLC-MS/MS in blood of the two cases[END_REF].

In our case, insulin glargine was identified and quantified using a high-resolution mass spectrometry method. This type of technology, unlike immunological methods, allows human insulin to be differentiated with certainty from all other analogs and to provide precise quantification on a wide range of biological matrices. Mass spectrometry is not affected by the problem of cross-reactivity and offers better specificity and sensitivity allowing the identification of multiple targets in a single test.

As the synthetic insulin analogs were developed with slight structural differences, their molecular characteristics remain very similar, especially in terms of molecular weight and structure. Consequently, they also have similar analytical characteristics which make their differentiation difficult even with the most modern analytical approaches. High-resolution mass spectrometry offers the possibility of separating interferences from the analyte and overcoming these limitations.

In the scientific literature, only Kim et al. [START_REF] Kim | Insulin glargine and insulin aspart overdose with pharmacokinetic analysis[END_REF] investigated insulin glargine in an overdose case by mass spectrometry. The authors conducted an analysis of the pharmacokinetics of this type of insulin by establishing insulin levels several times. Only the M1 metabolite of insulin glargine was identified and quantified 7 h (18 ng/mL), 30 h (19.5 ng/mL), 54 h (15.3 ng/mL), and 80 h (9.6 ng/mL) after injection.

The authors observed a multiphasic peak, probably due to the delayed release from the depot or the presence of several injection sites where the absorption rate was different.

Our results for insulin glargine cannot be compared with previous data as the parent compound was never identified after overdose. The therapeutic range for this type of insulin has not yet been defined, but according to a study by Bolli et al. [START_REF] Bolli | Plasma exposure to insulin glargine and its metabolites M1 and M2 after subcutaneous injection of therapeutic and supratherapeutic doses of glargine in subjects with type 1 diabetes[END_REF] when insulin glargine was subcutaneously administered at a therapeutic dose of 0.3 units/kg, the M1 metabolite, mostly responsible for the glucodynamic effects, was found at a maximum concentration of 470 pg/mL.

In this study, insulin glargine was never present in the blood circulation, irrespective of the injected dose. Kim et al. [START_REF] Kim | Insulin glargine and insulin aspart overdose with pharmacokinetic analysis[END_REF] also reported that insulin glargine is never found in circulation above the LOQ, unlike its major metabolite M1, suggesting that the parent molecule is not the main responsible for the hypoglycemic activity.

Danne et al. [START_REF] Danne | Insulin glargine metabolite 21(a) -Gly-human insulin (M1) is the principal component circulating in the plasma of young children with type 1 diabetes: results from the PRESCHOOL study[END_REF] again performed a study in 2012 involving a group of children under 6 years of age treated with insulin glargine.

They analyzed blood samples taken 24 h after subcutaneous injection of insulin glargine and only identified the M1 metabolite, which was listed as the main component mediating the hypoglycemic effect. The limitation of this study was that analysis was performed on specimens collected at +24 h, and therefore, the results do not necessarily reflect the values of the parent molecule at other times.

Since buying the reference standard of the M1 metabolite is very difficult (not proposed by any vendor), it was not possible to quantify it in our case. However, we investigated the M1 metabolite in our sample and the observed mass spectrum was compared with data in the literature. We have identified a peak whose spectrum corresponds to the fivefold ion charged m/z 1150.3317 described in the paper of Yong-Xi et al [START_REF] Kim | Insulin glargine and insulin aspart overdose with pharmacokinetic analysis[END_REF]. In our case, the fivefold charged ion was observed at m/z 1150.89968. The M2 metabolite was also investigated by comparing the mass spectrum with the literature data, but it was not found. This is in agreement with the study published by Bolli et al. [START_REF] Bolli | Plasma exposure to insulin glargine and its metabolites M1 and M2 after subcutaneous injection of therapeutic and supratherapeutic doses of glargine in subjects with type 1 diabetes[END_REF].

In order to be able to explain our findings, various hypotheses can be elaborated. First of all, according to the study by Bolli et al.

[3] insulin glargine, when detectable, appears very early in blood.

Therefore, one possibility could be that the time between injection and death was very short. As a hypoglycemic syndrome can last for many hours before causing death, this hypothesis is not plausible. In another study conducted by Agin et al. in 2007 [22], it was shown that insulin kinetic shows some inter-individual variability which affects the rate of transformation of insulin glargine into M1 metabolite. This variability could be explained by genetic differences in the activity of the carboxypeptidases responsible for its metabolism.

Indeed, in a study carried out on 69 individuals, the rate of conversion of glargine to M1 in 30 min varied between 46% and 98%.

Another hypothesis is that the injection route in our case was not the standard subcutaneous administration route, as this is the unique fatal case of intravenous insulin glargine overdose described in the literature. It is possible that the metabolism of insulin glargine is different when injected intravenously. In a study by Werner et al.

[23], the kinetics of insulin glargine was compared after intravenous and subcutaneous injection. When injected intravenously, insulin glargine accounts for 15%-37% of total circulating insulin while when injected subcutaneously, the parent molecule was almost never found. Thornton et al. in 2012 [START_REF] Thornton | Intravenous overdose of insulin glargine without prolonged hypoglycemic effects[END_REF] presented a case of a patient accidentally receiving 100 units of insulin glargine intravenously instead of subcutaneously. As the long-lasting properties of insulin glargine are due to its absorption kinetic, intravenously it acts like regular human insulin. In fact, the patient had an unremarkable course and recovered without any parenteral glucose. Insulin glargine dosage was not performed in this case.

Finally, as proposed by Werner et al. [START_REF] Werner | Equipotency of insulin glargine 300 and 100 U/mL with intravenous dosing but differential bioavailability with subcutaneous dosing in dogs[END_REF], since insulin glargine was injected at very high dosage, its presence can be explained by a possible saturation of the proteolytic enzymes responsible for the conversion of insulin glargine into its metabolite. This phenomenon was also observed in a study published by Lucidi et al. [START_REF] Lucidi | Glargine metabolism over 24 h following its subcutaneous injection in patients with type 2 diabetes mellitus: a doseresponse study[END_REF]. The authors evaluated the metabolism of insulin glargine in diabetic patients, observing that while the parent molecule was not detectable after a therapeutic dose injection, insulin glargine could be found after a slight overdose. These results suggest that at high dosages there is a greater opportunity to find the parent molecule.

As no injection site was found in our case but only a venous catheter at the left wrist (not submitted to analysis), the possibility of an intravenous administration cannot be eliminated.

| CON CLUS ION

The use of insulin in a forensic context is not new. Investigation In this article, the authors have presented a case of insulin glargine overdose. Insulin glargine has never been found in postmortem blood samples due to its rapid transformation into its major metabolite. Its presence at a toxic concentration could probably be due to the use of a route of administration different from the classic subcutaneous way, such as the intravenous route.

The metabolism of this substance could indeed be different when injected intravenously. As this represents the first case of a death reported in the literature with such high insulin glargine concentration in blood, it will be necessary to document further cases to confirm the hypotheses.

R E FE R E N C E S

15564029, 2023, 3 ,

 3 Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/1556-4029.15247 by Cochrane France, Wiley Online Library on [17/05/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License

  In the case described, neither the amount of insulin injected, nor the number of injection sites were known. After acute poisoning, there is a change in the pharmacokinetics of insulin glargine due to the slowed absorption from the injection site and the possibility of prolonged clearance. From a clinical point of view, identification of the insulin analog responsible for the overdose is essential. In most of the cases F I G U R E 1 Extracted chromatogram and spectra for insulin glargine from blood specimen by liquid chromatography coupled to highresolution mass spectrometry (LC-HRMS). Insulin glargine: m/z [M + 7H] 7+ 867.46288. Concentration was 1.06 mg/L.

F I G U R E 2

 2 Extracted chromatogram and spectra information for insulin glargine metabolite M1 (21A-Gly-insulin). M1: m/z [M + 5H] 5+ 1150.89969.

  of an insulin intoxication should include the assessment of various factors such as comprehensive site inspections and a full review of the medical history and subsequent biological analyses. However, toxicological tests are rarely performed. Insulin glargine is a longlasting insulin and overdose tends to cause delayed hypoglycemic episodes. Therefore, it seems essential to be able to identify and monitor concentrations in the clinical context in order to anticipate the type and duration of treatment. In a medico-legal situation, it is instead fundamental to identify the type of insulin used, which represents evidence of the administration of exogenous insulin. In this case, it has been demonstrated how modern analytical approaches such as high-resolution mass spectrometry allows to overcome the analytical issues related to this substance and to provide adequate evidence.

  1. Fédération Française des Diabétiques [French Federation of Diabetics]. La découverte de l'insuline à 100 ans! [The discovery of insulin at 100 years old!]. 2021 https://www.feder ation desdi abeti ques.org/feder ation/ actua lites/ 100-ans-de-la-decou verte -de-linsu line-decou vrez-lhist oire-et-les-comba ts-de-la-feder ation. Accessed 27 Dec 2021

15564029, 2023, 3, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/1556-4029.15247 by Cochrane France, Wiley Online Library on [17/05/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License


Nadja Groysbeck

Victor Hanss

Mariel Donzeau

Jean Marc Strub

Sarah Cianferani

Danièle Spehner

Mounib Bahri

Ovidiu Ersen

Mikhail Eltsov

Patrick Schultz

Jean-Marc Strub

Sarah Cianférani

Mikhael Eltsov

Guy Zuber
email: zuber@unistra.fr

M Eltsov

J.-M Strub

Bioactivated and PEG-Protected Circa 2 nm Gold Nanoparticles for in Cell Labelling and Cryo-Electron Microscopy

Keywords: bioconjugation, electroporation, gold labeling, gold particles, nuclear import, transduction

Introduction

Understanding the human cell architecture at the molecular level relies on the availability of advanced microscopes and labeling tools. Developments in cryo-Electron Microscopy (cryo-EM) now enable the recording of cellular images at nanometric resolution detailing multiple molecular components in near native frozen-hydrated conditions. [1] Large protein assemblies such as nuclear pores, ribosomes, microtubules or filaments can be readily visualized, whereas smaller components remain difficult to identify. [2] Gold nanoparticles (AuNPs) with an inner metallic core are popular EM labeling tags, as they efficiently scatter electrons and can be readily functionalized with antibodies to target selected cellular structures/proteins. In most cases, immunogold labeling for cellular EM imaging is performed after resin-embedding on sections of chemically cross-linked cells. However, the visualization of biomolecules in cryo-EM relies on the vitrification of living cells and on maintaining the native cell structure in a frozen-hydrated state throughout the sectioning or milling process up to image acquisition. The strict maintenance of the temperature below -140 °C does not allow for gold labeling following the classical process. This implies that labeling can only be performed before vitrification on unfixed living cells. This constraint has motivated a quest for new tags and methodologies. [3,4] Ideally, the labeling tool must be specific for its target, clearly recognized within the specimen and its use should not denature the specimen. In case intracellular proteins are targeted the probes must be able to freely diffuse in the cytosol.

We previously demonstrated that ultra-small 0.8 nm AuNPantibodies can be transported into living cells using a cationic lipid formulation and label their target, namely in this case, nuclear RNA polymerase II (PolII). [5] The size of gold nanoparticle attached to the probe was however too small to be revealed effectively by cryo-EM in frozen hydrated cell sections (data not shown). The linkage chemistry was furthermore poorly defined, potentially causing an uncertainty in the distance between the detected gold domain and the targeted epitope. We then screened for gold nanoparticles with increased diameters to facilitate their detection. Mercaptobenzoate-coordinated AuNPs of precise composition [6,7] appear extremely attractive in this regard. They can not only be produced with sizes between 1.2 to 3 nm [8] but also can be functionalized with antibodies via thiolate exchange [9] in aqueous phase. The thiolate-for-thiolate exchange tremendously simplifies the preparation of AuNP-conjugates and opens new ways to design advanced AuNP probes [10] for in situ labeling of supramolecular assemblies and cryo-EM analysis. [START_REF] Marjomaki | Proc. Natl. Acad. Sci[END_REF][START_REF] Martikainen | [END_REF][13][14] By following the synthesis protocol of thiobenzoate-coordinated gold nanoparticles reported by Levi-Kalisman, [7] we discovered that sodium borohydride reduction of gold(III) chloride in the presence of 5,5'-dithio-bis(2-nitrobenzoic acid) led to 1.4 nm AuNPs covered by a Au-S coordination shell consisting of thioaminobenzoates (TABs) and thionitrobenzoates (TNBs). [15] These mixed TAB-, TNB-coated AuNPs (AuZ) appeared extremely pertinent for bioconjugation purposes and intracytosolic live cell applications. First, the gold nanoparticles reacted well in water near pH 7 with excess thiolated peptides mainly by substitution of TNBs to peptides, producing a gold particle functionalized with 8-9 peptides and the zwitterionic TAB. Second, the organic shell did not dramatically alter HeLa cell viability after cytosolic delivery by electroporation and remained bound to the gold particles. [15] Indeed, the human cells rapidly recovered from the transient electric pulse-mediated permeabilization and withstood the cytoplasmic presence of peptide-functionalized AuNPs. The 1.4 nm AuNP probes coated with the capping peptide of sequence CALNNG [16] could diffuse freely in the cytoplasm and did not accumulate in clusters inside cells as was observed for 2 nm particles delivered by cationic lipid formulation [5] or penetrating peptides. [17] Only SV40 Nuclear Localization Signal (NLS) [18] conjugated nanoparticles were found to accumulate in the nucleus while Nuclear Export Signal (NES)-conjugated AuNPs were exported from the nucleus.

While these previously published data clearly demonstrated that AuZs are attractive platforms for biofunctionalization and production of AuNPs suitable for delivery into living cells, the 1.4 nm sized-particles still fall short for direct detection with the current electron-dose limitations of cryo-EM for biological specimen. [5,19] It was estimated that AuNPs larger than 2 nm are currently needed for direct detection in cryo-vitrified cell sections. [14] However, this size appears to represent a threshold for protein binding. [20] AuNPs below 2 nm display a strong curvature, and therefore the nanoparticle surface available for binding biomolecules by other means than coordination bonds is limited. [21] With increasing size, the AuNP curvature flattens and the exposed surface presents a larger contact area available for non-specific binding of biomolecules. [22] In the crowded cytosol, weak superficial interactions between AuNPs and the cellular components contribute more to slowing down of the diffusion of the particle than the increase of the hydrodynamic diameter associated with the larger size. [23,24] Hints of the toll taken by non-specific binding of 2 nm AuNPs to cellular components are images showing heterogeneous diffusion pattern and clustering of the nanomaterial in subcellular domains [17,25,26] as well as low specific activity when the AuNPs are equipped with functional elements targeting cytosolic enzymes. [27] Minimizing nonspecific binding of molecules using surface-protecting components such as high molecular weight polyethyleneglycol (PEG), which is widely used to decrease non-specific binding of nanomaterial to extracellular constituents [28,29] should enhance binding selectivity and improve the trafficking of the nanomaterial following cytosolic entry.

Here, 2 nm AuZs were synthesized and their surface was functionalized with SV40 NLS peptides and surface capping agents, by thiolate to thiolate exchange. Amongst the various tested capping elements, the 2 kDa polyethyleneglycols (PEG) was the most effective and was coordinated to the AuNP surface in combination with a NLS peptide at different proportion. After characterization, the functionalized AuNPs were first incubated with living HeLa cells to assay cytotoxicity and nonspecific cellular penetration and distribution. The gold nanoparticles were then delivered into the cytosol of living cells using an electroporation procedure and cell viability, probe distribution, and nuclear import efficiency were assayed. The PEG-,NLScoated AuNP (AuPEGNLS) with an equal amount of NLS and PEG conjugated to the AuNP surface showed weak non-specific binding to general cellular components while performing highly efficient NLS-mediated nuclear import. We further demonstrate that the 2 nm AuPEGNLSs after electroporation are directly detected within the nucleus in unstained vitreous cell sections by cryo-EM.

Results and Discussion

Synthesis and Characterization

The AuZs were synthesized in acetonitrile/water mixture [30] by sodium borohydride reduction of auric chloride in the presence of Ellman's reagent. [31] Characterization by gel electrophoresis, transmission electron microscopy, and MALDI-TOF revealed a monodisperse solution of particles with an average diameter of 2.02 nm (SD 0.21, n = 105) and a molecular mass of 60 kDa (Supporting InformationFigure S1, Supporting Information). The gold particles were then reacted with increasing molar ratios of the NLS peptide whose sequence (CALNNGAGP-KKKRKVED) contains the classical SV40 NLS [18] and with a previously described gold particle capping peptide whose sequence (CALNNG) [16] carries a reactive Cysteine at its Nterminus. The degree of ligand exchange on the particle surface was analyzed by monitoring stably bound NLS peptides to AuNPs by gel electrophoresis (Figure S2, Supporting Information). Substitution of AuZ-coordinated thiobenzoates by the NLS peptide expectedly increased when the peptide/AuZ ratios rose from 4 to 30. Excess NLS-coating of the 2 nm AuZ particles led to precipitation, which was not observed with the 1.4 nm AuZ. [15] This undesired precipitation is likely a consequence of the surface area of a 2 nm particle (calculated area of 12.6 nm 2 ) that is twice the one of a 1.4 nm particle (area of 6.15 nm 2 ) and prompted us to complement the conjugation of the bioactive NLS with a surface protecting element. Thiolated reagents ensuring effective protection for gold surfaces from associating with biomolecules are scarce. Glutathione [17,27] and 0.6 kDa thiolated PEG [32] were first tested as passivation agents for AuZ. Unfortunately, mixing the glutathione or the 0.6 kDa thiolated PEG with AuZ led to precipitates that were uneasy to re-suspend in aqueous solutions near pH 7.0. In contrast, addition of CALNNG peptide [16] or thiolated 2 kDa PEG to AuZ maintained the gold particles in suspension and formed the AuC and AuPEG components, respectively, via the expected thiolate to thiolate exchange. The NLS peptide was incorporated in the AuPEG shell at relative thiolate amounts of 11, 20, 33, 50, and 66%, thus yielding the AuPEGNLSs with the indicated NLS% (Figure 1A).

All functionalized AuNPs were purified by gel filtration chromatography, concentrated by ultracentrifugation, and behaved as stable colloids. Their electrophoretic mobility was different than the starting AuZ (Figure 1B). Regardless of the NLS proportion, the AuPEGNLS did not enter the gel, indicating that the thiolated 2 kDa PEG are coordinated onto the gold surface in a high density. The intensity of Coomassie blue staining of each AuNP grew as a function of increasing NLS proportion, indicating that the number of NLS peptides conjugated per AuNP increased as well (Figure S3, Supporting Information). All AuPEGNLSs show UV-vis spectrum similar to that of AuZ with a similar absorption shoulder at 520 nm (Figure 1C; Figure S4, Supporting Information). The concentration of the gold nanoparticles was determined by spectrometry measurement at 520 nm using an absorption coefficient of 510 000 M -1 cm -1 according to the formula proposed by Liu et al. [33] The MALDI-TOF Mass spectrum of the AuPEGNLS50% (Figure 1D) was representative of all the other AuPEGNLS spectra (Figures S5-S10, Supporting Information). A broad distribution of masses centered at ≈75 kDa was observed as compared to 60 kDa for AuZ. The roughly 15 kDa difference in mass likely measures the exchange of about ten 0.2 kDa TNBs to ten 2 kDa PEGs and 1.8 kDa NLSs. This

AuPEGNLS33%

AuPEGNLS50% AuPEGNLS66% AuPEGNLS20% B)The cell viability as estimated with the MTT assay. [34] Values are averages and standard deviations of quintuplicates. C)Light microscopy images and distribution profiles of AuNPs along the indicated lines. Cells were fixed with 2% glutaraldehyde, permeabilized with 0.1% saponin and the gold particles were silver-enhanced for detection.

substitution number suggests that the AuPEGNLS50% is grafted with 5 PEGs, 5 NLSs and residual TABs and TNBs. Electron micrographs showed evenly distributed individual particles on the carbon film. Statistical analysis of 41528 imaged particles resulted in an average diameter of 1.96 nm and a standard deviation of 0.38 nm.

Distribution Profiles Upon Addition to Living Cells

The above-characterized AuNPs were then incubated with living HeLa cells for 14 h in order to analyze their cytotoxicity (Figure 2). HeLa cells were freshly trypsinized to detach them from the Petri dishes, concentrated to obtain 10 5 cells in 10 μL of PBS and mixed . Cell suspensions were diluted in 1 mL cell culture medium containing 10% serum and AuNPs remaining in solution were separated from cells by centrifugation. The cell pellets were then re-suspended in cell culture medium for culture during 14 h. B)Cell viability was estimated with the MTT assay. [34] Values are averages and standard deviations of quintuplicates.

with 5 μM of each AuNP. After 10 min of incubation, the cells were diluted in cell medium containing serum and incubated for growth before analysis of cell viability and AuNP localization 14 h latter. HeLa cells with proteolytically removed adhesion receptors (e.g., trypsin cleaves superficial proteins involved in adhesion) but with native plasma membranes tolerate well the presence of the different AuNPs as evidenced by the recovery of <70% viable adhered cells relative to control even in the presence of 1 μM AuPEGNLS50%. (Figure 2B). The localization of the AuNPs was then monitored. The cell culture media containing unbound gold particles were removed. The cells were washed with PBS, fixed with glutaraldehyde and subjected to silver-enhancement (Figure 2C).

The AuZ particles gave rise to intense cytoplasmic aggregates likely because of their ability to react with thiols on the cell surface and their uptake into vesicular compartments. The AuC, AuPEG, AuPEGNLS 11% and AuPEGNLS 22% were barely seen on the cell surface and even less so within cells suggesting that they do not significantly bind to the plasma membrane (Figure 2C). Accumulation into discrete dots at the plasma membrane and within the cytoplasm was observed for AuPEGNLS33%, 50%, and 66%. The staining intensity increased as a function of the NLS content in the AuPEGNLS shell. The AuPEGNLS66% even strongly adhered to the glass support. The electrostatic associations between cationic NLSs and anionic proteoglycans present on the cell surface and the continuous recycling of the plasma membrane into intracellular vesicles may account for the observed gold particle distribution. [35] Regardless of the coverage, the AuNPs did not accumulate in the nucleus and did not diffuse into the cytosol. Intravesicular entrapment of gold particles without cytosolic release upon addition to living cells is expected [15,36] since the rupture of the endosomal bilayer, although possible, [37,38] remains a rare event.

Distribution Profiles after Entry into the Cytoplasm of Living Cells

The cytotoxicity and distribution of the AuNPs within living cells was investigated after having delivered the probe using a com-mercially available electroporation device [39] and electric pulses that were previously optimized for antibody transduction [15,40] (Figure 3 and Figure 4). AuNPs at the indicated final concentrations were incubated with freshly suspended HeLa cells in PBS. The solutions were then subjected to 3 electric pulses of 10 ms at 517 V cm -1 to create transient holes in the plasma membrane and hence cytosolic entry of the extracellular applied components. After the electric pulses, excess AuNPs in solution were removed and the electroporated cells were cultivated for 14 h before testing the recovery of cell viability and the distribution of AuNPs as described above. The cell viability was compared to cells in PBS without application of electric pulses or to cells in PBS treated with electric pulses. Except for AuZ, application of the 3 electric pulses and presence of AuNPs did not affect cell viability (Figure 3B). The highly thiol-reactive AuZ may trap essential cellular components and induce cell death.

The cellular distribution of the electroporated functionalized AuNPs (Figure 4A) was remarkably different than the ones reported in Figure 2C, confirming that the gold particles entered the cell and that their intracellular location was determined by the functions introduced on their outer shell. The nonfunctionalized AuZ did not diffuse freely but rather accumulated in cell membrane-bound aggregates or within intracellular vesicles.

The mobility of the thiol-reactive AuZ was probably hampered by its ability to react with thiolate-containing cell components and hence be trapped there. The internalized AuCs appeared to distribute throughout the cell, including to some extent in the nucleus. Aggregate formation was also clearly observed in perinuclear vesicles resembling lysosomal compartments. The AuPEGs appeared homogeneously dispersed in the cytoplasm and were mostly excluded from the cell nuclei in agreement with the fact that nuclear import of inert material larger than 5 nm in diameter is severely restricted by the nuclear pore complex. [41,42] In fact, the 2 kDa PEG molecules surrounding the 2 nm gold particles have a contour length of 12.5 nm which increases the maximal diameter of the particles to 27 nm. In contrast to the AuPEG, the NLS functionalized AuPEGNLSs accumulated in the nuclei and preferably in the nucleoplasm rather than in nucleoli. To analyze the nuclear accumulation of AuNPs, we plotted the intensity profile along a line crossing cytoplasmic and nuclear regions of the silver enhanced cells (Plots in Figure 4A). The intensity ratios between the average nuclear and cytoplasmic values were then calculated to quantify the nuclear accumulation (Figure 4B). The nuclear accumulation of the AuPEGNLSs consistently increased with the NLS density indicating that nuclear import depends on the NLS coverage. The maximum import was reached with 33% and 50% NLS coverage and was found to decrease for the 66% NLS coverage. This may be related to the intracellular aggregation observed in the control experiments with AuPEGNLS66% nanoparticles. Although the import efficiency was comparable for AuNPs with 33 and 50% coverage, the lower standard deviation of AuPEGNLS50% prompted us to employ them to prepare samples for EM observation. A first HAADF-STEM imaging of the AUPEGNLS50% particles delivered into living HeLa cells was performed at room temperature after silver enhancement and resin embedding (Figure 4C). With the exception of AuNPs entrapped within vesicular compartments at the vicinity of the nuclear envelope (black arrows), only a few particles were detected in the cytoplasm while the vast majority of AuNPs were found in the nucleus as single particles.

Distribution Profiles after Entry into the Cytoplasm of Synchronized Cells

To further demonstrate without ambiguity that the NLSfunctionalized AuPEGs are shuttled into the nucleus via the nuclear pores using the importin pathway and not trapped during mitosis, [43] DNA replication was inhibited with a double thymidine block to hold the cells in early S phase (Figure 5A). [44] The synchronized cells were then released from the DNA synthesis inhibition just before the electroporation procedure and the AuNP distribution was analyzed after growing the cells for 6.5 h (Figure 5B). At the arrest time, most synchronized cells remained in S phase and did not undergo nuclear envelope breakdown and mitosis. The distribution profiles of the different AuNPs were extremely similar to those obtained 14 h after cytosolic delivery and without thymidine block (Figure 4). The AuPEG, AuC, and Au-PEGNLS11% were mainly distributed in the cytosol with clear contact to the cytosolic face of the nuclear envelope. The AuPEG-NLS20% entered the nucleus albeit poorly. Darker grains near the nuclear envelope were observed, suggesting that these gold particles, 6.5 h after entry were still proceeding for association to importin and for passage across the gel protecting the hole of the nuclear pores. [41,45] The AuPEGNLS33% and 50% with the highest NLS density were efficiently imported into the nucleus with apparently few nanoparticles remaining in the cytosol. The AuPEG-NLS66% also accumulated in the nucleus but was also detected in perinuclear compartments resembling lysosomes, likely because the number of NLS peptides attached to these gold particles favor unspecific binding to the external surface of the plasma membrane and engulfment in intracellular vesicles via the plasma membrane recycling process. The living cells containing AuPEGNLS50% were then grown for 3,6,8,10, and 21 h in order to analyze the dynamics of intracellular AuNP distribution before and after mitosis (Figure 6).

The HeLa cells slowly formed adhesion contacts onto the cell culture dish and the AuPEGNLS50% readily accumulated in the cell nuclei following the electric pulses. Robust and near complete nuclear import was observed during the S phase when the nuclear envelope remains intact (3, 6, and 8 h time points). The cells then entered and exited mitosis without major perturbation, confirming that HeLa cells withstand the transient electric pulse-mediated permeabilization and the intracellular presence of AuPEGNLS50%. The fast nuclear import of the NLSequipped AuNPs following delivery parallels the behavior of NLScontaining proteins and confirms the relative inertness of the probes. [42] Of note, 2 nm AuPEGNLSs were also efficiently shuttled into the nuclei of U2OS following electroporation without marked cytotoxicity (Figure S11, Supporting Information), indicating that this protocol is applicable to other human cell lines.

Cryo-EM Imaging

In a final experiment, we investigated whether the 2 nm gold particles could be detected without silver enhancement within the unstained and fully hydrated cell nucleus by Cryo-Electron Microscopy Of Vitreous Sections (CEMOVIS). [1,46] The functionalized AuPEGNLS50% were delivered into the cytoplasm of living cells by electroporation and the cells were grown for 18 h before vitrification by high pressure freezing. Ribbons of vitreous cell sections were obtained using a cryo-ultramicrotome, collected on EM grids and imaged in cryo-EM (Figure 7).

At low magnification (Figure 7B), the cryo-TEM image showed a cell section centered on the nucleus and surrounded by the cytoplasm. The light grey holes correspond to transmitted information through the specimen only whereas the dark grey corresponds to an image of the specimen plus the carbon film. Magnification into a hole crossed by the nuclear envelope was performed to observe a nuclear pore and electron dense dot-like structures compatible with the size of AuNPs (Figure 7C). The contrast of these single dots was higher than for subcellular structures such as biological membranes but expectedly faint. To verify that these densities indeed represent the AuNPs, we applied a higher electron dose to the sections (120 e Å -2 ) than one normally used for imaging (10 e Å -2 ) (Figure 8). This treatment results in radiolysis of biological molecules but not of inorganic materials. The 2 nm-sized densities remained after radiolysis and became even more contrasted, thus confirming the presence and the ability to detect 2 nm gold particles in two-dimensional cryo-EM images of a crowded cellular section. All detected particles appeared as single and non-aggregated entities. Furthermore, and in agreement with our light microscopy results, a higher concentration of AuNPs was observed within the cell nucleus.

Conclusion

To use functionalized gold nanoparticles for intracellular labeling and cryo-EM observations, the penetration and diffusion abilities of the probe must be worked out in concert. We report the optimization of the surface coverage of 2 nm TAB-, TNB-protected AuNPs with bioactive NLS ligands and surface-protecting PEG in a controlled way through thiolate exchange. We then delivered these AuNPs into living cells by electroporation. The PEG coverage considerably limited intracytosolic clustering and nonspecific adherence to cellular components ensuring a robust SV40 NLS targeting of the nuclear import pathway and the shuttling of the gold particle into the nucleus. Direct detection of in-dividual 2 nm AuNPs within the nuclei of cryo-fixed cells, proves that 2 nm-sized AuNPs provide sufficient contrast to be detected even when embedded within the crowded environment of the cell nucleus. We strongly believe that this identified size range and PEG coverage opens multiple possibilities to generate specific gold immunolabelling tools for deciphering the distribution of intracellular components within living cells after delivery using a convenient electroporation procedure.

Experimental Section

General: Materials and additional protocols were described in the supporting information. The staining profile was measured using Image J [47] by plotting the gray value profiles underneath the indicated lines spanning the cell nuclei.

Synthesis of AuPEGNLS50%: A solution of 10 μM 2 nm AuZ [30] (90 μL, 0.9 nmol) was mixed with 1 M HEPES pH 7.4 (10 μL) and then rapidly added to a freshly mixed solution containing 10 mM CALNNGAGP-KKKRKVED peptide (4 μL, 40 nmol), 5 mM tris(-2-carboxyethyl)phosphine, pH 7.4 (8 μL, 40 nmol), and 20 mM thiolated PEG 2000 (PEGSH; 2 μL, 40 nmol) into a 0.5 mL polypropylene tube. The mixture was incubated at room temperature for 16 h. The crude mixture was then purified by size exclusion chromatography (Sephadex G-25, PBS as elution buffer) followed by 7 cycles of ultrafiltration (dilution in sterile PBS) using a 0.5 mL Microcon® centrifugal filter device with 30 kDa cut-off (Merck, Molsheim). At the end of the process, a 60 μL volume was recovered containing AuPEGNLS50% at an estimated concentration of 12 μM (0.72 nmol; 80% recovery yield). The other AuPEGNLSs at different NLS percentage were prepared using the same procedure and an identical [NLS thiolate peptide+PEGSH]/AuZ ratio of 80/0.9. After ultracentrifugation, the Au-PEGNLS50% was obtained at a concentration of 30 μM.

Synchronization of HeLa Cells: Adherent HeLa cells were synchronized using a double thymidine block method. [44] Thymidine (Sigma) was added to the cell culture media at a final concentration of 2 mM from a 100 mM stock solution in water. The cells were incubated for 16 h at 37 °C, after which the culture media was removed, the cells were washed three times with sterile PBS and normal growth medium was added again. Following 8 h at 37 °C, thymidine was added a second time at a final concentration of 2 mM for additional 16 h.

Electroporation Protocol: Transient permeabilization of the plasma membrane of living cells was conducted using the Neon R transfection system [39] with three 10 ms pulses at 517 V cm -1 . [40] For a final electroporation volume of 10 μL, HeLa cells (2 × 10 5 cells in 10 μL of the Neon Buffer R) were mixed with each functionalized gold nanoparticle (50 pmol at 5 μL of a 10 μM solution in PBS). After electroporation, the cells were diluted into prewarmed cell culture medium with 10% fetal calf serum (FCS) but without antibiotics (1 mL). The cells were pelleted by gentle centrifugation (100 RCF), suspended into warmed cell culture medium with 10%FCS without antibiotics (0.5 mL) and added into 24-well plates containing 13 mm diameter cover glass or into 35 mm Ibidi dishes (Ibidi: ref 80 136 or 80 156). Plates or dishes were then placed inside a cell culture incubator at 37 °C, 5% CO 2 for cell adherence and growth.

Cell Viability Assay: The assay was done according to a published procedure. [34] Electroporated cells and untreated cells were diluted in 0.5 mL cell culture medium and divided in fractions of 100 μL in a 96well plate. The cells were incubated at 37 °C, 5% CO 2 , for 24 h before addition of thiazolyl blue tetrazolium bromide solution in PBS (10 μL of a 5 mg mL -1 ) for quantification of the released formazan.

Cellular Specimen preparation for Light Microscopy: The treated cells, grown on cover glasses or Ibidi dishes, were washed twice in PBS (2×1 mL). The cells were then fixed with 2.0% glutaraldehyde in 100 mM Sorenson's buffer (1 mL) for 4 h. After removal of the fixative and 3 PBS washes (1 mL), unreacted aldehydes were blocked by incubation with a 100 mM Sorenson's buffer, pH 7.4 containing 50 mM glycine (1 mL, 20 min). The plasma membrane was permeabilized using 0.1 m Sorenson's buffer pH 7.4 containing 0.1% (w/v) saponin (1 mL, 15 min) before proceeding for silver-mediated amplification of the gold particles according to a modified Danscher protocol using silver acetate and propyl gallate. [48] Briefly, the phosphate-buffered solution was replaced by a 0.1 m citrate solution, pH 6.7 containing 2% sucrose (5 washes). Silver-enhancement of the specimen was then done in a dark room for 8 min using a freshly prepared 6 mM silver acetate solution in 0.16 m sodium citrate, pH 6.7 containing 2 mM propyl gallate and 20% gum arabic. The silver-mediated AuNP staining solution was then removed from the specimens with extensive washes using 0.16 m sodium citrate solution, pH 6.7 (1 mL) and then PBS (0.5 mL). The specimens on the cover glasses were finally mounted onto 3×1 inch microscope slides (knittelglass.com) using Fluoromount-G (Southern Biotech, Ref. 0100-01, batch I2819-WC79B). Observations were carried with Leica DM5500B microscope equipped with HCX PL Apo 63×1.40 oil PH3CS objective and a Leica DFC350FX camera. When cells were grown on 35 mm Ibidi dishes, the observations were carried out with an Olympus IX83 equipped with LUCPLFLN 40×0.6 objective and a CMOS Hamamatsu Orca Fusion camera (C144440-20UP) directly after the silver-enhanced procedure.

Figure 1 .

 1 Figure 1. Preparation and characterization of polyethyleneglycol -, Nuclear Localization Signal peptide -decorated AuNPs. A)Illustration of the AuNPs and synthesis scheme. The percentage refers to the relative amounts of NLS peptide to total (PEG + NLS) thiolates. As a control, the AuZwas also reacted with CALNNG forming the AuC nanoparticles. B)Polyacrylamide gel electrophoresis analysis of the starting AuZ, purified AuC, and AuPEGNLS at the indicated NLS proportion. C)UV-vis spectrometry of AuZ (in red) and AuPEGNLS50% (in blue). D)MALTI-TOF Mass spectrum of the AuPEGNLS50%. E)Electron microscopy image of AuPEGNLS50% and population analysis of the gold particle diameter.

Figure 2 .

 2 Figure2. Analysis of cell viability and gold particle distribution following their incubation with HeLa cells for 14 h as indicated. A)Freshly trypsinized HeLa cells (10 5 cells/10 μL PBS) alone or with 5 μM of each AuNP (10 μM AuPEGNLS50% in the * marked condition), were incubated for 10 min before dilution in 1 mL cell culture medium containing 10% serum. Cells were then dispatched in plates for cell culture at 37 °C during 14 h (final concentration of AuNPs after dilution: 0.5 μM (1 μM for*). B)The cell viability as estimated with the MTT assay.[34] Values are averages and standard deviations of quintuplicates. C)Light microscopy images and distribution profiles of AuNPs along the indicated lines. Cells were fixed with 2% glutaraldehyde, permeabilized with 0.1% saponin and the gold particles were silver-enhanced for detection.

Figure 3 .

 3 Figure 3. Analysis of cell viability following electroporation of HeLa cells incubated with the indicated gold particles. A)Freshly trypsinized HeLa cells (10 5 cells/10 μL PBS) alone (-) or with 5 μM of each AuNP (10 μM AuPEGNLS50% in the * marked condition) were subjected to 3 electric pulses of 10 ms at 517 V cm -1. Cell suspensions were diluted in 1 mL cell culture medium containing 10% serum and AuNPs remaining in solution were separated from cells by centrifugation. The cell pellets were then re-suspended in cell culture medium for culture during 14 h. B)Cell viability was estimated with the MTT assay.[34] Values are averages and standard deviations of quintuplicates.

Figure 4 .

 4 Figure 4. Intracellular AuNP distribution after AuNP delivery by electroporation. A)Light microscopy images and distribution profiles of the indicated AuNPs 14 h after transient plasma membrane permeabilization, and subsequently silver enhanced cells. The graphs represent intensity plots along the indicated lines and reveal the AuNP distribution. B)Quantification of nuclear accumulation.Differences in staining between the nucleus and the cytosol were measured using gray plots from silver-enhanced cell images containing the indicated delivered AuNPs. Values are averages and standard deviations of 14 measures. C)HAADF-STEM images of a HeLa section containing delivered AuPEGNLS50%. After delivery and cell culture, the cells were fixed, the AuNPs were enhanced with silver and the specimen embedded in resin for room temperature electron microscopy imaging. For sake of clarity, the nuclear envelope (dotted red line) and the barely visible AuNPs (orange dots) were marked. The 3 black arrows point to AuNPs entrapped in cytoplasmic vesicles.

Figure 5 .

 5 Figure5. Analysis of cell content 6.5 h after application of electric pulses to HeLa cells synchronized to be in early S phase and incubated with the indicated AuNPs. A)Scheme of the procedure. B)Distribution of the indicated gold nanoparticles in synchronized HeLa cells 6.5 h after release of the inhibition of DNA replication and application of the electric pulses (3 pulses of 10 ms at 517 V cm -1 ). The cells were fixed with 2% glutaraldehyde, permeabilized with 0.1% saponin and the gold particles were enhanced with silver for detection by light microscopy.

Figure 6 .

 6 Figure 6. Light microscopy images of HeLa cells containing AuPEGNLS50% at different time points after electroporation and release of the inhibition of DNA replication. The cells were fixed with 2% glutaraldehyde, permeabilized with 0.1% saponin and the gold particles were silver-enhanced for detection by light microscopy.

Figure 7 .

 7 Figure 7. Cryo-EM workflow of high-pressure frozen HeLa cells electroporated with AuNLSPEG50%. A)The vitrified specimen was sectioned in cryogenic conditions using a cryo-ultramicrotome. The resulting translucent ribbon was deposited on a copper grid covered with a perforated carbon film and observed in cryo-Electron Microscopy. B)Cryo-EM micrograph showing a portion of cell centered on its nucleus where the nuclear envelope is highlighted. C)Magnified area around the nuclear envelope (black rectangle in (B)) showing subcellular structures including a nuclear pore (white arrow) separating the cytoplasm (left side) from the nucleus (right side). Gold particles in small boxesare enlarged 4 times (large inserts with 2nm black scale bars on bottom left corners) for better visibility.

Figure 8 .

 8 Figure 8. Identification of gold particles in the nucleoplasm after radiolysis of biological structures by irradiation with a high electron dose. The structural aspect of the vitreous section gradually changes after irradiation by 20, 80, 120e -Å -2 (upper row). Gold particles are the only high contrast structural features remaining after high dose irradiation.

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Small Methods 2023, 2300098 © 2023 The Authors. Small Methods published by Wiley-VCH GmbH 2300098 (8 of 12) 23669608, 0, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/smtd.202300098 by Cochrane France, Wiley Online Library on [17/05/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License

Acknowledgements

This research was supported by the ITI Innovec (IdEx (ANR-10-IDEX-0002), SFRI (ANR-20-SFRI-0012)), the French Infrastructure for Integrated Structural Biology (FRISBI ANR-10-INBS-05), and the ITMO Cancer (Col-orME, 22P096-00). N.G. received a Ph.D. fellowship from the IdEX Unistra (Université de Strasbourg and Investissements d'Avenir). V.H. received a fellowship from the CNRS. Robert Drillien styleedited the text.

Data Availability Statement

The data that support the findings of this study are available in the supplementary material of this article.

Supporting Information

Supporting Information is available from the Wiley Online Library or from the author.

Conflict of Interest

The authors declare no conflict of interest.