Anthracycline drugs, including doxorubicin (DOX), epirubicin (EPI), daunorubicin (DAU), and idarubicin (IDA), with lipophilicity P from 0.8 to 15, were investigated. We have found that in addition to hydrogen-bonded supramolecular ensemble formation with n = 24, there are two other competing contributions that enable increasing n under strong polar interactions (APO(DOX)) or under strong hydrophobic interactions (APO(IDA) of the highest efficacy). The encapsulation/release processes were investigated using UV-Vis, fluorescence, circular dichroism, and FTIR spectroscopies. The in vitro cytotoxicity/growth inhibition tests and flow cytometry corroborate high apoptotic activity of APO(drugs) against targeted MDA-MB-231 adenocarcinoma and HeLa cells, and low activity against healthy MCF10A cells, demonstrating targeting ability of nanodrugs. A model for molecular interactions between anthracyclines and APO nanocarriers was developed, and the relationships derived compared with experimental results.The interactions of chemotherapeutic drugs with nanocage protein apoferritin (APO) are the key features in the effective encapsulation and release of highly toxic drugs in APO-based controlled drug delivery systems. The encapsulation enables mitigating the drugs’ side effects, collateral damage to healthy cells, and adverse immune reactions. Herein, the interactions of anthracycline drugs with APO were studied to assess the effect of drug lipophilicity on their encapsulation excess  Recent advances in cancer treatment, achieved through the development of precision guided surgical techniques, novel drugs, and modern technologies for cancer detection and monitoring ,2,3,4, hThe interactions of ANTR derivatives with APO nanocages are the key features for drug loading and transporting through the biological medium. However, the lack of systematic investigations of these interactions prevents successful development of APO-based drug delivery systems. In this work, we propose a novel comprehensive physicochemical model of these interactions showing non-trivial dependence of the drug loading on various types of interactions, including hydrogen bonding, polar interactions, hydrophobic interactions, and supramolecular complex formation, in addition to the usually considered simple entrapment of drugs in solution-filled APO nanocages. The mathematical modelling presented should be useful to gain better understanding of not only the drug binding but also the drug releasing processes, induced by pH change and APO opening in the intracellular environment of a cancer tissue.In vitro testing of the activity of APO-encapsulated anthracyclines, including doxorubicin (DOX), epirubicin (EPI), daunorubicin (DAU), and idarubicin (IDA), widely differing in their lipophilicities, was performed using flow cytometry and cytotoxicity/growth inhibition methods for HeLa cervical cancer and MDA-MB-231 adenocarcinoma breast cancer cells, as well as non-tumorigenic MCF10A epithelial cells.The nanocarrier-based CDDs can provide significant benefits to anticancer therapy, including targeted drug delivery (TDD) ,14,15, pThe CDD system may also serve as an efficient vehicle to overcome MDR ,7,10, whDifferent kinds of nanoparticle (NP) nanocarriers have been tested in preclinical studies, but only a few of them have been so far clinically approved because of concerns with toxicity, immunogenicity, or sequestration by the reticuloendothelial system . On the Anthracyclines, investigated in this work, are antibiotics that inhibit DNA and RNA synthesis by intercalating into the nucleic acid duplex ,51, damaP = 0.8 to 15.0. We hypothesize that, owing to the richness of binding sites on the inside surface of the protein cavity, APO can bind, via supramolecular interactions, drug molecules with different lipophilicities and enable efficient encapsulation of the drugs for targeted delivery. While doxorubicin has been extensively studied before /[APO] was found to be close to 31.5, indicating that a saturation of nanocages was achieved. Therefore, for further studies, the molar ratio of drug:APO = 100:1 was selected for all drugs under study to minimize the amount of the drugs used. The encapsulation molar ratios for different ANTR derivatives in APO nanocages are presented in To determine the optimum molar concentration ratio of drug:APO for the encapsulation solution, the tests were performed using doxorubicin as the model anticancer drug, using the ratios of DOX:APO from 50:1 to 200:1. For the ratios from 100:1 to 200:1, the encapsulation molar ratio 3 at carbon C4. For the rest of the anthracyclines investigated, the encapsulation ratios are decreasing from 31.5 to 29.3 with increasing drug lipophilicity from 0.8 to 3.5. For this group of anthracyclines , the observed encapsulation ratios follow the decrease in dipolar interactions between the drugs and APO with increasing drug lipophilicity.The highest encapsulation ratio was observed for IDA (33.6) which also shows the highest lipophilicity of 15.0, mainly due to the absence of group -O-CHε280 = 468,000 cm−1 M−1), whereas the spectra of anthracyclines show absorption bands P1 and P3. With increasing concentrations of APO, the intensities of all three peaks increase linearly. As seen in the spectra for APO(IDA) nanocarriers in As seen in CAPO confirms that no aggregation of APO(drug) nanocarriers occurs.These data indicate that the interactions of DOX and IDA with APO nanocages differ significantly. The linear dependence of the intensity of P2 on APO concentration To obtain further information concerning the interactions of anthracycline drugs with APO nanoshells, we have applied circular dichroism (CD) spectroscopy which enables evaluation of conformation changes in the APO protein during the encapsulation of ANTRs. Two anthracycline drugs with the largest difference in their lipophilicity were taken for comparison. The CD spectra were recorded in the far-UV spectral region from 190 to 240 nm A. The spThe percentage of α-helix for APO alone was predicted to be 92.6%. After the encapsulation of anthracyclines, the spectra of APO(IDA) and APO(DOX) revealed deviations in both minima and maximum from their original pattern, indicating that the α-helix content in the APO structure has changed. For APO(IDA), the percentage of α-helix decreased and was predicted to be 89.9%, whereas for the APO(DOX), it slightly increased to reach 93.2%. Thus, IDA with its stronger hydrophobic properties disrupts the α-helical APO structure, while DOX, with more pronounced dipolar interactions, stabilizes the APO α-helix.2 groups on the APO surface. The attached folate moiety binds to a folate receptor (FR) exposed in cancer cell membranes. The best-known isoform, FRα, is a glycosyl phosphatidylinositol-anchored glycoprotein. It is overexpressed by cancer cells. Upon binding the folate-conjugated nanocarriers, it can internalize them through the receptor-mediated endocytosis [Modifications of the outer surface of APO nanocages with a gold film and functional ligands have recently been proposed . Herein,ocytosis .λem = 593 nm. This phenomenon is likely due to the quenching of DOX fluorescence by FA, recently documented by Husseini et al. [To confirm the attachment of FA to the APO(drug) surface, detailed measurements using FTIR, UV-Vis, and fluorescence spectroscopies, with an APO(DOX) as the model nanocarrier have been performed. As seen in i et al.  and Santλmax = 280 nm remained unchanged (−1 and a band at 1485 cm−1 for stretching vibration of phenyl ring of FA (Further proof of the FA attachment to the nanocarriers was obtained from FTIR spectra for APO(DOX)@FA showing a band for stretching vibration of carbonyl group (–C=O) of the attached FA at 1697 cmThe drug release experiments indicate that the FA attachment to APO(DOX) surface has no significant influence on the stability of APO(DOX) nanocarriers.Since the primary mechanism of chemotherapeutic drug action is based on the induction of apoptosis, we have investigated how the APO nanocages with encapsulated ANTR drugs fare relative to free drugs. In our experiments, the MDA-MB-231 cancer cells were treated with free ANTRs  at concentrations: 1, 10, 100, and 1000 nM, or different drug-loaded APO nanocages (APO(DOX), APO(EPI), APO(IDA)) containing equivalent amounts of drugs. After 72 h of treatment, the cells were analyzed, and the percentage of apoptotic cells determined by flow cytometry assessment of Annexin-V-stained cells . We haveAmong the anthracyclines examined, free IDA shows the strongest apoptotic action toward MDA-MB-231 cancer cells, with the percentage of late apoptotic cells approaching 89.1 ± 3.6%, and the APO(IDA) nanocarriers also show a high efficiency of 81.6 ± 3.3%  nanocarriers was further confirmed using a cytotoxicity/growth inhibition assay. Again, APO(IDA) has shown the highest activity and induced more apoptotic MDA-MB-231 cancer cells than the APO nanocarriers loaded with other anthracyclines. Therefore, for further experiments, we selected APO(IDA) to evaluate the effectiveness of APO nanocarriers in treatment of cancer cells: HeLa and MDA-MB-231, in relation to that of healthy MCF10A cells.In The treatment of cancer cells with the 1.0 µM IDA in APO(IDA) nanocages resulted in the death of 86 ± 3% and 93 ± 2% of MDA-MB-231 adenocarcinoma cells and HeLa cervical cancer cells, respectively, compared to 59 ± 8% dead cells observed for MCF10A healthy cells D.It follows from these data that the encapsulation of idarubicin in apoferritin cavity protects healthy cells against the harmful effects of the drug. Thus, by using APO nanocarriers, the anthracycline chemotherapeutic drugs can be delivered to cancer cells in a targeted manner.50 values for free ANTR drugs and APO-encapsulated nanodrugs have been calculated from the dose–response curves and are presented in The dose–response relationships for free ANTR drugs and APO-encapsulated ANTR nanodrugs have been evaluated based on the flow cytometry data obtained for MDA-MB-231 breast cancer cells treated with these drugs and analyzed by the annexin V and 7-ADD staining. The drug dose–response curves are presented in The APO shell nanocarriers offer a unique ability to safely transport highly toxic chemotherapeutic drugs to the targeted tumors while enabling straightforward drug loading and unloading operations. They effectively prevent damage to healthy cells, mitigate the drugs’ side effects, and protect drugs against deactivation by the biological environment. Moreover, the APO nanocarriers do not induce any immunoresponse of the organism. In our experiments, we have demonstrated that chemotherapeutic drugs from the anthracycline group can be encapsulated in APO nanocarriers with high efficiency despite considerable differences in drug lipophilicity ranging from 0.8 to 15. Based on the experiments performed, we have encountered the following mechanisms of encapsulation of these drugs, showing different dependences of drug binding ability on their lipophilicity:P = 0.8 to 3.5), the efficiency of overall binding of drugs to the APO nanocarriers decreases with increasing drug lipophilicity, following the trend for polar interactions between the drugs and APO. This trend is consistent with the behavior of DOX, EPI, and DAU.(i) For ANTR drugs with low lipophilicity (P = 3.5 to 15), where the polar interactions play a reduced role, an opposite trend is observed with the overall drug binding efficiency increasing with the increasing lipophilicity. This trend is characteristic for hydrophobic interactions between the drugs and a matrix and it is consistent with the behavior of DAU and IDA. The interactions of APO with IDA, showing the highest lipophilicity (P = 15.0), are the strongest among the drugs examined. This coincides with IDA’s highest encapsulation ratio. Although the hydrogen bonding could play a role here, the detailed analysis of the number of possible hydrogen bonds, the anthracycline derivatives can form with APO, indicates that all the drugs under study can form up to 8 or 9 hydrogen bonds. Therefore, the likely reason for the increased binding ability of IDA are the hydrophobic interactions with the inner APO surface. This type of interaction of IDA may be enhanced because of the protrusions of aromatic rings of amino acids to the inner space of APO cage, for such amino acids as phenylalanine (Phe), tryptophan (Trp), and tyrosine (Tyr). The hydrophobicity of these amino acids increases in order: Tyr < Trp < Phe, according to the Wimley–White interfacial hydrophobicity scale for amino acids [(ii) For ANTR drugs with high lipophilicity (no acids  that comno acids . They man) with n = 24 ± 3. Unlike in the case of cisplatin binding to APO, with the formation of APO(cisplatinn) complex, where the metal atom actually interacts with nitrogen free electron pair of His 133 of APO, the interactions of anthracyclines with APO are due to the supramolecular interactions involving multiple hydrogen bonds at the biorecognition site for ANTR triple ring on the inner APO surface. The ANTR drugs can form up to nine hydrogen bonds with APO. The molecular ratio of APO:drug = 1:24 can be compared with the theoretical values for non-specific close-packing adsorption of a model ANTR derivative, DOX, on the inner APO surface, ranging from 150 to 308 DOX molecules per APO nanocage, depending on the drug molecule orientation ((iii) The interactions (i) and (ii) are superimposed on lipophilicity-independent specific interactions of anthracyclines with binding sites on the inner surface of APO, referred to by Kilic et al.  as the centation . On the (iv) A considerable number of drug molecules can also be bound to the external surface of an APO. For instance, in the case of a non-specific adsorption of DOX in side-on orientation, the maximum surface excess of DOX is found to be 693 molec/APO on the outer APO surface, compared to 308 molec/APO on the inner APO surface . HoweverKd = 1 ÷ 10 nM), to absorb folate-coated APO nanocarriers via receptor-mediated endocytosis [Targeting of tumor cells has been achieved through the known ability of APO to recognize the receptors: TfR1 and SCARA5, which are overexpressed in rapidly proliferating cells, such as cancer cells ,36, and ocytosis ,14.In vitro evaluation of the APO(drug) activity in MCF10A, MDA-MB-231, and HeLa cells, using flow cytometry and cytotoxicity/growth inhibition assays, performed in this work, show a reduced drug delivery rate with APO(drug) nanodrugs in comparison to the free drugs. This is expected due to the slow APO nanocage opening during the drug release phase. These measurements also corroborate the high apoptotic activity of the APO(ANTR) nanodrugs against cancer cells: MDA-MB-231 adenocarcinoma and HeLa cervical cancer cells, with APO(IDA) showing the highest efficacy by generating 82% apoptotic cells in MDA-MB-231 cell line at 1 µM IDA encapsulated in APO(IDA). This contrasts with lower apoptotic activity of the nanodrugs against healthy MCF10A epithelial cells, demonstrating selective cytotoxicity against targeted cancer cells. This means that folate-enhanced APO(ANTR) nanocarriers offer targeted drug delivery and can mitigate drug side effects, reduce damage to healthy cells, and hold a potential to overcome multiple drug resistance.(1)0pol due to polar interactions leading to the formation of hydrogen bonds and immobilization of ANTR molecules on the inner surface of an APO cage on hydrophilic sites;∆G(2)0hph due to hydrophobic interactions leading to the immobilization of ANTR on the inner APO cage surface on hydrophobic sites;∆G(3)0ens due to the supramolecular ensemble formation APO(ANTRp), with the maximum encapsulation number P = nens,max = 24;∆G(4)0trap due to the entrapment of free ANTR drug molecules in the inner volume of an APO nanocage;∆G(5)0el due to electrostatic attraction of positively charged ANTR molecules, at pH < pKa of the protonation of the -NH2 moiety, and negatively charged dissociated local carboxyl groups of APO. The point of zero charge (pzc) for APO is: pzc = 4.4 [∆Gzc = 4.4 ,62). TheBased on the discussion presented in the previous section, we have developed a model for anthracycline drugs encapsulation in APO nanocages, based on the Gibbs free energies for the following interactions:0trap = 0, unless a mechanism for linking ANTR molecules together is provided, e.g., by bridging via metal ions. The encapsulation number n is defined here as the total number of drug molecules bound to the inner APO surface and trapped in the inner space of the APO cage.The total Gibbs free energy for ANTR encapsulation is given by:24) are filled before other sites available on APO surface are filled in processes (1) and (2). The contribution of the entrapment (4) to the encapsulation under conditions of our experiments, is less than 1 drug molecule per APO nanocage. It has to be emphasized that a considerable increase of the drug concentration in the encapsulation solution, to increase the drug entrapment rate, is limited due to the relatively low solubility of the ANTR derivatives. The increased entrapment can in principle be achieved by providing a mechanism for linking ANTR molecules together in the APO inner solution, for instance by Fe(III)-bridging [ntot are:Among the processes listed above, process (3) has recently been recognized and well described in the literature . Due to bridging . An attebridging . UnfortuThe model calculations for competing processes (1) and (2) are described below.(1)Binding of ANTR drugs on non-hydrophobic sites of APO.APO,1 can be expressed by the reaction:CD is the drug concentration in solution, CAPO is the concentration of APO nanocarriers in solution, n1 is the number of drug molecules already bound on hydrophobic sites of the inner APO surface, and n1,max is the maximum number of available non-hydrophobic sites for drug binding per APO molecule. The binding equilibrium constant K1 is given by:The immobilization of a drug molecule D on a free non-hydrophobic binding site on the inner APO surface □n1,max and K1 depend on P. Since n1,max evidently decreases with increasing lipophilicity P, we assume a simple relationship:a1 < 0. By combining Equations (4) and (6), after some algebra, one obtains:In these equations, P. We assume that the effect of P on K1 follows a linear dependence since P is a simple distribution ratio of the drug between a hydrophobic (octanol) and a hydrophilic (aqueous PBS buffer) phase:b1 < 0. Therefore, the equation for n1 becomes:Due to the low internal surface coverage of APO by the drug molecules, the lateral interactions between immobilized drug molecules are expected to be rather weak, especially since the number of drug molecules bound to hydrophilic sites on APO decreases with increasing P given by the equation:P, using:(i)P and the number of residual sites n1,res, which remain available even at the highest P due to the characteristic properties of the ANTR triple ring structure, ora hyperbolic averaging for the number of sites lost with increasing (ii)P, according to the phenomenological equation:a loss function representing the number of blocked hydrophilic sites, which increases with increasing lipophilicity Kbl is the ratio of the number of blocked sites to the number of free hydrophilic sites and P = 0.However, this function has a singularity at Kbl represents the process of hydrophilic sites blocking and its value is associated with increasing lipophilicity P of the ANTR derivatives. We assume a simple relation: Kbl= m1P, where m1 > 0 since blocking increases with lipophilicity. Thus:The constant P increases from 0 to infinity. After a simple transformation, including reflection of the loss function with respect to the abscissae and shifting upwards by n1 to the encapsulation excess as a function of P:It is seen from this equation that n1 on P, showing a decrease in dipolar interactions and H-bonding with increasing lipophilicity is plotted in This dependence of the encapsulation excess For the limiting conditions, we have:P, the approach (i) fails at P values close to 0, showing errors that depend on the parameters involved. Therefore, approach (ii), based on the loss function for the hydrophilic sites, has been used in further analysis.While both approaches, (i) and (ii), lead to satisfactory results for intermediate and large values of (2)Binding of ANTR drugs on hydrophobic sites of APOWhile the anthracycline drugs with high lipophilicity still contain the basic triple ring with many of the original polar functional groups, found in DOX, and are still able to form H-bonding and undergo polar interactions, the dominating contribution to the ANTR binding to APO nanocages is now due to the hydrophobic interactions APO-ANTR. These interactions increase with increasing lipophilicity, as expected, whereas the dipolar interactions and hydrogen bonding are being diminished toward the minimum required by the structural definition of anthracyclines family.K2 is given by:For hydrophobic binding, expressed by the reaction:n2,max, increases linearly with increasing lipophilicity:We assume that the maximum number of available sites for binding, a2 is now positive since more hydrophobic sites appear in drug molecules with increasing P. By combining Equations (19) and (21), one obtains:Therefore, in contrast to Equation (5), K2 also depends on P. We assume a simple linear dependence:b2 > 0. Therefore, the equation for n2 becomes:a2P and b2P are positive and there is no singularity in the range of P accessible experimentally, i.e., from P = 0 to 15. The contribution n2 to n, given by this equation, is plotted in The value of For limiting conditions, we have:n1 and n2 is represented in The sum of contributions The fitting performed provides the following values of constants:P changing from 0 to 15, close to the maximum monolayer coverage of ANTR drugs per inner APO surface in vertical orientation: 308.3 − 24 = 284.3 (see: With the parameters shown in Equations (24) and (25), the number of hydrophobic sites spans from 0 to 283.5 per APO, for 4.3 see: .The experiments performed indicate clearly that the activity of ANTR derivatives, encapsulated in APO nanocage carriers , is lower than that of free ANTR drugs. This behavior is expected due to relatively slow drug release from the nanocarriers in comparison to the immediate drug availability in the case of free drugs. This is corroborated by monitoring of the drug release from APO nanocages. In fact, the drug release transients, recorded for APO(ANTR) nanocarriers for all ANTR drugs under study in buffer solutions with pH: 7.4, 5.6, 4.6, and 3.6 SI, , clearlyThe advantage of using APO-based drug nanocarriers for targeted CDD is their ability to recognize cancer cells and deliver the drug payload selectively to cancer cells. The APO nanocarriers, utilized in this work can directly interact with the TfR1 and SCARA 5 receptors ,36 overeThe drug release transients, presented in 50 values for the nanodrugs. The cytotoxicity tests have demonstrated the high activity of APO-encapsulated ANTR nanodrugs toward the cancer cell lines and support the proposed model of ANTR drugs encapsulation. This model is based on evaluation of the effects of different kinds of interactions that influence the encapsulation excess of ANTR drugs in APO nanocage nanocarriers. The proposed encapsulation model may serve not only to assess the in vitro activity of the APO-based nanodrugs but can also help in studying other novel theranostic drug nanocarriers. The use of nanocarriers is now becoming a necessity due to the impasse in further development of systemic chemotherapies due to adverse side effects and collateral damage to healthy cells, often leading to the organ failure and even death. We hope that the model proposed here can be in future adopted also to other drug nanocarriers and can serve in the development of controlled drug delivery systems for successful cancer treatment.In summary, an extensive cytotoxicity testing has been performed to evaluate the in vitro activity of novel anthraquinone nanodrugs toward cancer cell lines , as well as the non-tumorigenic cell line . The characterization of drug activity included determination of the dose–response curves for free ANTR drugs and APO-encapsulated nanodrugs for MDA-MB-231 cells, flow cytometry tests to determine the extent of apoptosis induced by the nanodrugs under study, cytotoxicity/growth inhibition tests, as well as the determination of EC2HPO4, 137 mM NaCl, 2.7 mM KCl, and 10 mM KH2PO4. McIlvain buffer (MI buffer) solutions  were prepared from 0.2 M Na2HPO4 and 0.1 M citric acid stock solutions. All aqueous solutions were prepared with deionized water (resistivity of 18.2 MΩ cm) purified with a Milli-Q reagent grade water system .The anticancer drugs, epirubicin (EPI), idarubicin (IDA), and daunorubicin (DAU), were received from Selleckchem , and doxorubicin (DOX) was purchased from Sigma-Aldrich Co. ; the drugs were used as received. The apoferritin from equine spleen (APO) was purchased from Sigma-Aldrich Co. . N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (EDC), N-hydroxysuccinimide (NHS), folic acid, and other chemicals were obtained from Aldrich Chemical Co.  and were of analytical grade and used as received. Phosphate-buffered saline (PBS) pH 7.4 consisted of 1.8 mM NaThe fluorescence spectra were recorded using LS55 Spectrometer  equipped with 20 kW Xenon light source operating in 8 μs pulsing mode. Separate monochromators for the incident and detector beams enabled use of monochromatic radiation with wavelengths from 200 to 700 nm. The dual detector system consisted of a photomultiplier tube (PMT) and an avalanche photodiode. The UV–Vis spectra were recorded using a Varian Cary 50 Bio UV–Visible Spectrophotometer  in the range from 200 nm to 700 nm at room temperature. FTIR spectra of APO nanocages were obtained using Model Nicolet iS-10 FTIR instrument  working in specular mode enabling the reflection from the sensor surface to be analyzed. The circular dichroism (CD) spectra were recorded on a J-715 spectropolarimeter . Far-UV (190–240 nm) recordings were performed in a 0.5 cm pathlength quartz cuvette.50 values for free drugs and APO-encapsulated nanodrugs were also obtained using ORIGIN software package.The model calculations of contributions of different kinds of interactions between APO nanocage carriers and ANTR drugs were performed using digital simulation and fitting utilities in Microsoft EXCEL software and ORIGIN v. 9.1 data analysis software . All statistical functions were calculated using ORIGIN. The dose–response relations and ECa values are close to 8.46 and 7.34, due to the amine group protonation and phenolic dissociation, respectively. Hence, the net charges carried by these anthracycline molecules do not differ significantly. However, the small structural differences, including additional -O-CH3 group at C4, as well as -CH2OH or -CH3 group at C13, result in considerable differences in drugs lipophilicity and supramolecular interactions with apoferritin protein, thus influencing the encapsulation efficiency. The measurements performed in this work were designed to evaluate the efficiency of encapsulation of anthracyclines under study in APO nanocages.The four anthracyclines studied have a similar structure and their main pKn = 5). For all experiments performed in this work, freshly prepared APO(ANTR) nanodrugs were used.The encapsulation of anthracycline drugs in APO nanocages was carried out by the disassembly/reassembly protocols based on those developed recently ,40,43,443. To activate carboxyl groups of the folic acid, 10 µL of FA solution was added to 900 µL of EDC/NHS mixture containing 7 mM EDC and 7 mM NHS in water and the resulting solution was allowed to react at room temperature for 30 min. The activated mixture was added to the APO(drug) nanocages, prepared earlier by centrifugation using Amicon Ultra-0.5 mL 3K  to remove water, and incubated overnight at room temperature. The APO(drug)@FA nanocarriers were purified through the Amicon Ultra-0.5 mL 30K and washed 3 times with Milli-Q water. The obtained product was dispersed in water for further characterization and application.To improve the effectiveness of APO(drug) nanocages in targeted drug delivery, we applied covalent binding of folic acid (FA) to an APO(drug) nanocage using a modification of the standard EDC/NHS reaction. We dissolved 10 mM FA in 5% NaHCOλex = 480 nm. All the data are expressed as the average of at least three determinations.The quantification of encapsulated anthracyclines was performed by determining the concentration of free drugs in solution, separated from APO(drug) NPs by filtration and the initial drug concentration in the encapsulation solution. The concentrations of free drugs were evaluated using fluorescence emission measurements, for The kinetics of drug release from APO(drug) was evaluated using PBS buffer pH 7.4 and McIlvain buffers with pH 3.6, 4.6, and 5.6. In brief, 300 µL of the appropriate buffer was added to the APO(drug) and samples were incubated for 6 h at 37 °C. At the pre-set time points (1 h apart), the solution with unbound drug was separated by filtration using Amicon-Ultra-0.5 mL 3K at RT. The container with APO(drug) nanocarriers was replenished with an equal volume of blank buffer (300 µL). The release study was carried out in triplicate for each pH.2. The cell lines used in this study: standard HeLa cancer cells, MDA-MB-231 human mammary gland breast cancer adenocarcinoma cells, and MCF10A human mammary non-tumorigenic epithelial cells, were obtained from American Type Culture Collection . HeLa and MDA-MB-231 cells were cultured as monolayers in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS). The growth medium for MCF10A cells contained DMEM, horse serum (5%), epidermal growth factor , hydrocortisone (0.5 mg/mL), cholera toxin (100 ng/mL), and insulin (10 μg/mL). Cells were routinely passed at 80–90% confluence, using trypsin/EDTA.Cells were cultured in a humidified incubator at 37 °C with 5% COTo determine the fraction of dead and apoptotic cells induced by targeted free anthracyclines  and APO nanocages uploaded with encapsulated anthracyclines , the Annexin V method was used. MDA-MB-231 cells were exposed to the indicated concentrations of anticancer drugs and APO nanocages for 72 h. Media and cells were then collected, pelleted, and processed according to the Muse™ Cell Analyzer Annexin V and Dead Cell Kit instructions (MCH100105). Briefly, 100 µL of cells in suspension were added to tubes. Next, 100 μL of the Muse™ Annexin V & Dead Cell Reagent was added to each tube. Samples were stained for 20 min at room temperature in the dark. Approximately 5000 cells were gated for analysis per sample; triplicate biological replicates were analyzed.2 atmosphere at 37 °C for 72 h to allow for colony formation and growth. After the 72-h treatment, pictures of each well on the six-well plates were taken. The cells were then fixed in 3.7% formaldehyde for 15 min. The fixed cells were rinsed with PBS and stained for 10 min using 0.1% crystal violet. After staining, the plates were rinsed with water to remove the staining solution and allowed to air dry. To quantify, the cells were solubilized in 1% SDS and the absorbance at 590 nm was measured. The percentage of cell survival (PCS) was calculated as follows:Cytotoxicity/growth inhibition assay is an in vitro cell survival assay based on the ability of a single cell to grow into a colony. HeLa, MDA-MB-231, and MCF10A cells were seeded onto six-well plates at a concentration of 150,000 cells/well such that they were 40% confluent at 24 h. The cells were then treated with IDA and APO(IDA) at concentrations: 0.0, 0.5, and 1.0 µM of the drug. After the treatment, cells were incubated in 5% CO


Prenatal exposure to mercury, stress, and depression may have adverse effects on birth outcomes. Little is known on the influence of chemical and non-chemical stressors on birth outcomes in the country of Suriname. We assessed the influence of prenatal exposure to mercury, perceived stress, and depression on adverse birth outcomes in 1143 pregnant Surinamese women who participated in the Caribbean Consortium for Research in Environmental and Occupational Health-MeKiTamara prospective cohort study. Associations between mercury , probable depression , high perceived stress , and adverse birth outcomes (low birthweight (<2500 g), preterm birth (<37 completed weeks of gestation), and low Apgar score (<7 at 5 min)) were assessed using bivariate and multivariate logistic regressions. Prevalence of elevated mercury levels, high perceived stress, and probable depression were 37.5%, 27.2%, and 22.4%, respectively. Mercury exposure was significantly associated with preterm birth in the overall study cohort  and perceived stress with a low Apgar score . Depression was not associated with any birth outcomes. These findings can inform policy- and practice-oriented solutions to improve maternal and child health in Suriname. Maternal exposure to chemical and non-chemical stressors may result in adverse health effects for mother and child. Exposures to mercury, stress, or depression that may result in adverse birth outcomes and long-term neurodevelopmental effects in children are of particular concern. Women prenatally exposed to mercury are more likely to deliver infants who have a low birthweight or who are small-for-gestational age , as wellThere is accumulating evidence that combined exposure to chemical and non-chemical stressors has a synergistic effect on maternal and child health ,11,12. AConsequences of adverse birth outcomes include mortality, delay in neurodevelopment for infants and children, and emotional and economic costs for families . Adversen = 5371 live births) indicated a neonatal mortality rate of 12.9%. Most of these neonates had a birthweight of less than 2500 g (71.2%) and were born prematurely (67.7%) [Suriname is a middle-income country situated on the northeastern coast of South America. The majority of its 541,638 inhabitants (66%) reside in urban areas, and the remainder in rural areas (21%) and the Amazonian interior rainforest (13%) . Surinam (67.7%) .−1 and the US EPA standard for human consumption of 1 μg g−1 in collected piscivorous fish  [In Suriname, mercury is mainly used in artisanal and small-scale goldmining in the Amazonian interior. Elemental mercury used in goldmining pollutes freshwater bodies where it ultimately accumulates in fish, the main protein source for the interior population. A study in Suriname found mercury levels above the European Union standard for human consumption of 0.5 μg g aimara) . High le aimara) . In addi aimara) . As therFindings of the Caribbean Consortium for Research in Environmental and Occupational Health (CCREOH)-MeKiTamara study, an ongoing prospective cohort study focused on maternal and child health in Suriname, showed that prenatal depression and perceived stress are important health issues among pregnant women in Suriname. The prevalence of high perceived stress in participants during the first two trimesters of pregnancy was 27.2%, while probable depression was 22.4% during the first or second trimester. Women experiencing high stress levels during the first two trimesters had almost twice the odds of having probable depression during the third trimester. Lower educational level and age ≥20 years) were significantly associated with probable depression during the third trimester [ years weDespite the potentially high levels of exposure to mercury, perceived stress, and depression and the high incidence of low birthweight and preterm birth in Suriname, no studies have examined the influence of chemical and non-chemical stressors on birth outcomes. As pregnant women are typically exposed to both kinds of stressors, it is important to not only examine the separate effects of the exposures, but also the combined effect on birth outcomes. The aim of this study is to examine the influence of perceived stress, depression, and mercury on birth outcomes in Suriname. Towards this end, the relation between hair mercury levels, the Cohen Perceived Stress Scale, and the Edinburgh Depression Scale, and the birth outcomes of low birthweight, low Apgar score, and preterm birth are assessed in the MeKiTamara cohort.The MeKiTamara study is a prospective environmental epidemiologic cohort study. MekiTamara means “creating a mother and child’s tomorrow” in Sranang tongo, Suriname’s lingua franca. Pregnant women were recruited in three regions of Suriname: the capital city of Paramaribo, the agricultural district of Nickerie, and the Amazonian interior.From December 2016 to July 2019, eligible pregnant women were recruited from (1) Paramaribo, at four hospitals  and at prenatal clinics and midwife facilities of the regional health department; (2) Nickerie, at the Mungra Medical Centre Hospital and at regional health department clinics and facilities; and (3) the Amazonian interior, at multiple health care clinics of the Medical Mission Primary Health Care Suriname. Women were eligible if they were 16 years or older, spoke Dutch, Saramaccan, or Trio, had a singleton gestation, were planning to give birth at one of the study sites, and provided written informed consent/assent. A total of 1143 women were included in this study.Interviewer-assisted surveys (including standardized questionnaires and a series of demographic questions) were conducted by trained recruiters using encrypted iPads. Survey data were uploaded to Research Electronic Data Capture (REDCap). REDCap is a secure web application for building and monitoring online surveys and databases, and can be used online or offline to collect data for research . The staQuestionnaires included Cohen’s Perceived Stress Scale and the Edinburgh Depression Scale. Hair was sampled late in the first trimester or early in the second trimester by cutting 1.5 g of hair as close as possible to the scalp using sterilized scissors. Samples were immediately stored in a sealed plastic pouch and refrigerated. They were then sent for storage at 4 °C until analysis at the National Zoological Collection of Suriname/Center for Environmental Research Lab at the Anton de Kom University of Suriname. Selected hair samples were evaluated at two laboratories and went through extensive quality assurance/quality control to ensure that the mercury being measured had been deposited within the hair shaft matrix and was not an external contaminant .Demographic variables were categorized into the following groups: age , marital status , household income ), educational level , race/ethnicity with African descent , Asian descent , or other ), parity , and region: urban , rural , and the interior .p < 0.001) [The total hair mercury concentration was reported in units of μg/g. The cut-off for elevated mercury levels was set at ≥1.1 μg/g according to the US Environmental Protection Agency (US EPA) action level. In addition, mercury levels were divided into quartiles. Hair mercury levels and blood mercury levels for this population were highly correlated  . Hair me< 0.001) . In addi< 0.001) .The Cohen Perceived Stress Scale (PSS-10) includes 10 items about the degree of experiencing stress due to having no control over things, nervousness, and not feeling confident about their ability to cope with things during the past month. There are five response options: 0 for never, 1 for almost never, 2 for sometimes, 3 for fairly often, and 4 for very often. The total score ranges from 0 to 40 points, 0 indicates the lowest stress level and 40 the highest stress level. In this study, the cut-off point was set at the 75th percentile 20) for a positively skewed distribution. Since the majority of the scores in the study population were low, we compared the top 25% with the rest of the data. In our earlier study of the MeKiTamara cohort, 27.2% of the 1143 pregnant women scored between 20 and 40 on the Cohen’s Perceived Stress Scale during the first or second trimester, indicating high perceived stress levels [ for a poThe Edinburgh Postnatal Depression Scale assesses postnatal depression, but has been validated for use prenatally. During the prenatal phase, it is known as the Edinburgh Depression Scale (EDS) . The EDSInformation was obtained from labor and delivery books completed by midwives in the hospitals, prenatal clinics, or midwife facilities where the baby was born. Low birthweight was defined as birthweight <2500 g, preterm birth as birth before 37 completed weeks of gestation, and low Apgar score as <7 at 5 min.p-value ≤ 0.05 was considered significant.Total mercury analysis was conducted with cold vapor atomic absorption spectrometry (CVAAS) . Questionnaire and demographic data were analyzed using SPSS version 20 . Bivariate logistic regression was used to determine the association between demographic factors and the exposure variables and between demographic variables and the birth outcomes. Bivariate logistic regression analysis was also conducted to determine the association between the exposure variables and LBW, PTB, and low Apgar score. We examined the exposure variables as both continuous and categorical variables. Since the results were similar for both types of variables, we decided to use categorical variables for ease of presentation and interpretability. Multivariable logistic regressions, adjusted for covariates that were significantly associated with both the exposure variables and with the birth outcome in question, were conducted. Interaction models were performed to examine the combined association between high mercury levels and high stress/depression levels on birth outcomes. A This study was approved by the Institutional Review Board (IRB) of Tulane University and the Medical Ethical Commission of Suriname’s Ministry of Health (VG 023-14). All included women (18+) provided written informed consent, and assent was obtained from women who were 16 or 17 years old.Demographic characteristics are presented in p = 0.097) and depression (p = 0.355) were not significantly associated with mercury levels. In a previous study, stress and depression were positively associated in this cohort [s cohort .p = 0.05). Neither depression nor high mercury levels showed an association with low birthweight, preterm birth, or low Apgar score (The average birthweight of the infants was 3014.51 g (SD 584.70), the average gestational age at delivery was 38 weeks (SD 2.44), and the average Apgar score was 9.45 (SD 1.64). Perceived stress was not significantly associated with low birthweight  or preterm birth , but it was associated with a low Apgar score; women with high perceived stress levels had a twofold higher risk of having a baby with a low Apgar score  with PTB in the overall study population  (p = 0.004) with a low Apgar score  , lower educated , and who had no previous live births  had a higher risk of having an infant with a low birthweight than women aged 20–34 years, higher educated, and with a history of ≥2 births . In the 05–5.83)  and perc3–46.70) . There wOver one out of three pregnant Surinamese women enrolled in the MeKiTamara study had elevated hair mercury levels, one out of four had high perceived stress levels, and one out of five had probable depression as defined by the EDS scale during early pregnancy. Hair mercury levels (first versus fourth quartile) were significantly associated with preterm birth. Older age (35+), primiparity, and lower educational level were significantly associated with low birthweight. Women with high perceived stress had almost tenfold higher odds of an infant with a low Apgar score.We found a statistically significant association between mercury exposure and preterm birth in the overall study cohort. This is in line with another study that showed a significant relation of high hair mercury levels of pregnant women with preterm birth . We did In the MeKiTamara participants, we see an extremely high likelihood (OR 47.76) of elevated hair mercury levels in women from the interior compared with women from rural areas. Our results also showed an increased risk (OR 2.51) for high mercury levels in the “other” ethnicity group. After splitting the “other” group into the sub-groups, we noticed that 54% of the participants were Indigenous women, of which, 62% had mercury levels in the fourth quartile. Further, 94% of the Indigenous women with elevated mercury levels were living in the interior of Suriname. After limiting our analysis to the interior participant subcohort only, we did not see any significant association between mercury levels and adverse birth outcomes. A reason for this may be the fact that the majority of the participants had elevated mercury levels, and the group with low exposure was too small to see an effect. It is known that mercury is used for artisanal goldmining in the interior, and that the Indigenous Peoples mainly subsist on fish consumption . Fish coWe found perceived stress to be significantly associated with a low 5-min Apgar score. A study which included women from urban and rural health centers in Iran, and using the PSS-14 scale to assess perceived stress, showed similar results. Women who perceived high levels of stress, and concern about feelings and interpersonal relationships had significantly higher scores for perceived stress which was associated with lower Apgar scores . As our Furthermore, our results showed that older age 35+), primiparity, and lower educational level were significantly positively associated with low birthweight. A systematic review of 41 studies found similar results regarding the association between primiparity and low birthweight . Infants+, primipWe saw a greater risk of low birthweight for women with lower education. As education and income are associated with each other and almost 70% of our participants had lower household incomes, this may be an explanation of the higher risks of low birthweight in lower-educated women. Further, their nutritional status may contribute to low birthweight, assuming that women with a low education and income are less knowledgeable about and maybe cannot afford to buy healthy food. This is confirmed by a study in South Carolina which showed an association between stress and low birthweight, modified by neighborhood, with infants of high poverty neighborhoods being more at risk for low birthweight .We did not find an association between depression and low birthweight, preterm birth, and low Apgar score. A review study looking at the association between prenatal depression and low birthweight and preterm birth between 1977 and 2013 reported less than a quarter of the 50 studies looking at preterm birth and depression to be significantly associated, and more than half of the 33 studies assessing the association between (low) birthweight to be significantly associated with prenatal depression [To our knowledge, this is the first study in Suriname that assesses the influence of non-chemical chemical stressors on birth outcomes, and the first to assess the combined effect of both chemical and non-chemical stressors on birth outcomes. Our adequate sample size, the even representation of the different ethnicities in Suriname, the three recruitment regions, and the range of socio-demographic factors enhance the generalizability to the Surinamese population. Although the questionnaires used in this study were not specifically validated for Suriname, they are standardized and have been used in other LMICs. Further, as perceived stress and probable depression were measured with a scale and not assessed by a mental health specialist, our results do not equate to a clinical diagnosis. However, because the EDS does not contain somatic symptoms and is validated for use during pregnancy, it is a robust scale and does not produce artificially high scores . FinallyOur results suggest that while mercury was not significantly associated with birthweight and low Apgar score, it may affect preterm birth. This gives more support to the idea that the Surinamese government should strengthen and enforce laws regarding mercury use, and ensure adequate monitoring of mercury use in goldmining to protect interior inhabitants, especially pregnant women and their infants. The population should also be well-informed of the adverse health effects of mercury exposure during pregnancy on mother and child. A recommendation would be to provide information about mercury levels in fish and the recommended levels of fish consumption during pregnancy. Since we did not examine fish intake, nor the levels of omega 3-fatty acids for the consumed fish in this study, we recommend further research. Regarding the high levels of perceived stress and depression as measured by questionnaires, our results call for effective and timely clinical prenatal screening by general practitioners, gynecologists, or midwives at prenatal visits. As this is the first study examining the influence of chemical and non-chemical stressors in Suriname, this research makes a significant contribution to the knowledge about maternal and child health in the country. Further research is needed to examine the association between mercury, depression, and birth outcomes. In addition, other risk factors for adverse birth outcomes such as substance use, nutritional status, chronic diseases, and prenatal care utilization should be considered. It is important to keep in mind that this study did not assess the association of prenatal mercury exposure with neurodevelopment outcomes in children in the cohort: this research is ongoing.


A small percentage of women choose to This study used a Grounded Theory methodology. Data from 13 women choosing homebirth and 15 choosing freebirth were collected between 2010 and 2014 and analysed over this time.birth outside the system. The basic social process, which explains the journey women took as they pursued the best and safest, was ‘finding a better way’. Women who gave birth outside the system in Australia had the countercultural belief that their knowledge about what was best and safest had greater authority than the socially accepted experts in maternity care. The women did not believe the rhetoric about the safety of hospitals and considered a biomedical approach towards birth to be the riskier birth option compared to giving birth outside the system. Previous birth experiences taught the women that hospital care was emotionally unsafe and that there was a possibility of further trauma if they returned to hospital. Giving birth outside the system presented the women with what they believed to be the opportunity to experience the best and safest circumstances for themselves and their babies.The core category was ‘wanting the best and safest,’ which describes what motivated the women to Shortfalls in the Australian maternity care system is the major contributing factor to women’s choice to give birth outside the system. Systematic improvements should prioritise humanising maternity care and the expansion of birth options which prioritise midwifery-led care for women of all risk. The majority of births (97%) in Australia occur in hospitals, 1.8% of women give birth in birth centres and 0.3% at home , howeverThe phenomenon of women choosing to freebirth or have a homebirth with risk factors homebirth has been explored by national and international researchers –14. ThesA previous traumatic birth experience is another major driver , 13, 17.Globally, childbirth occurs within predominantly biomedical models of care , 21 and birth outside the system – that is, to have a midwife attended homebirth despite identified complexities, or a freebirth where birth at home is planned to be intentionally unattended by health professionals.The aim of this study was to explore what motivates Australian women to what motivates women to birth outside the system?’This research was conducted between 2010 and 2014. Grounded Theory was the most suitable research method for this study, because it is ideal in circumstances where relatively little is known about a subject . GroundeBirks and Mills (2011) suggest ten essential elements that should be used in the research process if a Grounded Theory is to be produced. These are: initial coding and catergorisation of data, concurrent generation or collection of data and analysis, writing memos, theoretical sampling, constant comparative analysis, theoretical sensitivity, intermediate coding, identifying a core category, advanced coding and theoretical integration and generating a theory. The application of Birks and Mills’ (2011) suggestion of adhering to the ten essential elements was adopted in this study , 29. TheThe process of selecting research participants for Grounded Theory research relies on the use of ‘theoretical sampling,’ , where tIn the beginning of the recruitment process, two women known to the authors were purposefully approached to participate in this research; one had had a freebirth and the other a birth at home with risk factors. Both consented to being interviewed and from there ‘snowball sampling’ was used, thus, study participants recommend future participants. Another valuable exercise during the recruitment process was to announce the research at the Homebirth Australia Conference in 2010 as this generated a lot of interest and many women and midwives offered contact information as potential participants. These volunteers were carefully screened against the selection criteria before being included in the study. As the study progressed, participants were theoretically sampled based on personal information they gave during recruitment. Charmaz (2006), suggests that theoretical sampling only becomes of value once your categories have been developed, as this enables the researcher to confirm and clarify these categories.We planned to recruit women who had experienced or were planning a freebirth or a homebirth with risk factors.Have had a freebirth or homebirth with risk factors in the pastAre pregnant and are planning a freebirth or homebirth with risk factorsAre intending to have a freebirth or homebirth with risk factors in the futureThe inclusion criteria for the participants were as follows:birth outside the system was therefore irrelevant. In total, the stories of 28 women were included as data for the study, 13 who had chosen homebirth and 15 choosing freebirth.Participants were included if their intention was to birth outside the system, regardless if they ultimately gave birth elsewhere. The criteria were defined as such because the interest was in what motivated a woman’s choice; whether or not the woman actually achieved a National Review of Maternity Services in Australia (NMR)  birth caused myself and my family significant distress and trauma for a long time afterwards’ (FB10). The presence of long-term effects after a traumatic birth left some participants questioning whether they would ever recover, with comments such as ‘I’ve never been the same since’ (HB11) and ‘it would change our family forever’ (FB11).birth outside the system. The participants believed that childbirth imprints on one’s life, is a normal process and needs ideal circumstances to work best. With the understanding that childbirth imprints on one’s life, participants pursued birth outside the system in the hope that the imprinting would be positive not negative. The belief that childbirth is a normal bodily function, predisposed participants to question the need for hospitalisation. Finally, the participants reported a detailed list of circumstances that they believe are required to ensure birth works best. These included: adequate hormonal function, an optimal environment, privacy and relaxation, active birth positioning and good physical, emotional and mental preparation. Based on their previous experiences, the participants believed that the hospital was not capable of facilitating the circumstances that birth needed to work best and was therefore incapable of providing the best and safest.The participants’ philosophical standpoint on childbirth played a particular role in their choice to birth outside the system, so too did their perspectives on risk. The participants’ perceptions of risk were that birth always entails an element of risk, the hospital is not the safest place to have a baby, and that interference is a risk. The participants also believed that their chosen place of birth may not mitigate the risk inherent in birth. As one woman put it,‘I always knew that there was no guarantee that the baby would be born alive or that it would live beyond the birth, but I think there is no guarantee with that in a hospital setting either’. (FB06).Just as the participants’ perspectives on childbirth informed their choice to ‘Automatically walking into a hospital I’m exposed to hospital bugs, that to me is unsafe ... a neonate’s immune system is not fully developed, I don’t want my babies exposed to that, I don’t even want myself exposed to that. So, they can’t possibly offer me a safe birth’. (FB05).They believed that the hospital is not the safest place to have a baby, citing additional and unique risks associated with this option. One woman commented:'If you can stay away from the hospital system, then you can minimise the amount of interference. I look at interference a bit like risk, like every time someone new comes across you or does something that’s a risk that something goes wrong, every time you get a medication there is a risk it’s the wrong one, every time they do something, there is a risk that flows onto something else, so if no one is doing anything to you or giving you any drugs or performing any unnecessary tests, then there is no risk there'. (HB05).Finally, the participants perceived that interference in the birth process is a risk. One woman commented:Because the participants wanted the best and safest for themselves and their babies, they sought out birth options that would limit risk. Hospital was perceived to be riskier, and the most likely site where birth would be interfered with, so it was discounted as a suitable birth option.Participants’ previous experiences with the hospital system, paired with their beliefs on childbirth and risk, led them to assume that the hospital could not provide the best or the safest, because they could not cater to their personal criteria by which ‘safest’ and ‘best’ were judged. The participants cited multiple reasons why the hospital was incapable of providing the best or safest and these have been categorised under the headings, ‘not enough resources to cope with demand’, ‘the environment was not like home’, ‘it’s like a cattle yard’, ‘staff are bound by hospital policy’, ‘they intervene’, ‘they fear birth’, ‘there would be tension around my autonomy’ and ‘hospital management of birth is emotionally unsafe’.One woman felt that the hospital system was inherently flawed, stating:'I’m not sure they really could have done anything better for me just because of the mentality ... it’s a revolving door and they’ve got to get this baby out the quickest way possible, whichever way suits us and then get this baby fed, don’t care how just get it fed and then get you out the door. I don’t know if they could have done anything better'. (HB07).Worse still, many women found hospital care traumatising, with one woman explaining,'I decided that should I find myself unable to access a midwife, I would birth at home – alone. Nothing that can happen to me or my baby at home could be much worse than what my second baby and I experienced in hospital. I will never subject myself, my baby or my family to such an ugly, traumatic and dehumanising experience again'. (FB1).birth outside the system.The participants concluded that the hospital could not provide the best or the safest, and so they set out on the journey towards ‘finding a better way’, which ultimately led them to birth outside the system, the participants described making a considered decision after first exploring other birthing options. This process followed a typical path, with the women ‘discovering that there are multiple birth options’. They move on to ‘meet with a variety of care providers’ and then ‘weigh it all up.’ After making the decision to circumvent the system, the women ‘get informed about out-of-the-system birthing options,’ which for some leads to homebirth and for others ‘forces them to consider freebirth.’In explaining their choice to ‘I just became more informed about my other choices ... [and this] just blew open a whole new world for me around another choice’ (HB04).As the women investigated, they discovered many birthing options of which they had hitherto been unaware. In her pursuit of a better way, one participant said,‘I googled everything and anything that I could get my hands on…read Ina May Gaskins stuff and yeah so got a lot more informed about the alternatives’ (HB06).As the women met with care providers and weighed up all their options, they came to a realisation that giving birth in hospital would not cater to their desire for the best and safest. It was from this point women described immersing themselves in information about homebirth and freebirth; one woman explains:For the women who chose homebirth with risk factors, their process of discovery of birth options stopped here; they hired a midwife and followed through with their midwife-assisted homebirth. For those who chose freebirth, the process of considering their options continued. In their quest to find a better way, some participants came to point where they were forced to consider freebirth. This was because other birthing options became either unavailable or unacceptable to them.All but one of the women reported having made contact with a midwife or care provider in order to discuss their birth options. The one woman who did not make contact had completed a PhD on the topic of medicalisation of childbirth and during that process had connected with women who described their choice to freebirth. Prior to becoming pregnant, this woman decided she would freebirth and thus did not engage the counsel of a care provider in the pursuit of her plans to freebirth.While the women choosing homebirth with risk factors chose to continue being cared for by a midwife and women who chose freebirth disengaged from this care, the two choices to give birth outside the system have their roots in a fundamental rejection of how maternity care is provided within the system. These two outside-the-system birth choices are united in their counter-cultural rejection of the care that is offered to women in mainstream maternity care services in Australia.'Yeah, like, if that [a midwife] had been available, I would have been quite happy to have the midwife help me in my home have my baby, I never would have considered unassisted ... I mean like I said, I never would have chosen to go down that path had the decision – I kind of felt like the decision was made for me, by denying me that choice'. (FB03).The majority of participants reported a preference to have a midwife in attendance, but ultimately freebirthed because they felt they had no other option, and so it was the best and safest option available to them at the time. One participant explained:In the process of finding a better way to birth, the participants realised that their chosen option subverted biomedical models of childbirth, and that they would have to formulate a strategy in order to manage opposition. The women anticipated that managing opposition within a hospital setting would be hard work, and they did not want to have face this task while in labour. As one woman put it, ‘I felt like it would be a constant struggle, my partner and I against the hospital staff’ (HB04). Another participant felt that managing opposition ‘seems like a lot of energy’ (HB06) to waste whilst trying to give birth.The strategies employed in order to manage perceived opposition included: arming themselves with knowledge so they could effectively defend their choice, strategic engagement of care providers – avoiding those who would oppose their choice and engaging those who would facilitate it, selective disclosure of their plans to avoid conflict, having people on their side to help advocate for their choice, and ‘playing the game,’ which involved bartering with and manipulating the system to ultimately get what they wanted without having to compromise on the best and safest.'I really feel like setting the scene for the freebirth for me was all about taking good care of myself and I invest a lot of time and money into having really good health care and I take really good care of myself'. (FB06).The participants made their birth choices based on their desire for risk reduction, therefore, they sought to mitigate the risk by focusing on their mental and physical preparation in order to experience the most optimal birth outcomes. One woman explained:They also gathered knowledge, skills and supplies to ensure that they felt adequately prepared to mitigate their unique risks, this preparation differed between women who chose freebirth and homebirth. The women who chose freebirth read about what they might need – as one participant noted, ‘we did a lot of research into what we would need to have the birth at home’ (FB01). Women who freebirthed collected equipment for resuscitation – ‘we had the little resuscitation kit’ (FB01) – and also equipment that would be required for an uncomplicated birth, ‘like sterilised scissors’ (FB05) to cut the cord after the birth. While, women who had hired a midwife did not make mention of specific items that they gathered in preparation for their homebirth, the women who freebirthed felt obliged to gather this equipment since they were taking full responsibility over what supplies would be available to them for their birth.'... everything from, if my waters break and there is staining in the meconium we are off to hospital, if you know, if I’m feeling unwell – you know we went through a – I listed all the situations with my husband and I sort of said if this happens, then we need to transfer to hospital, if that happens then we need to transfer to hospital'. (FB08).They also planned for all possibilities, so that they were clear in their minds about what plan of action would be taken in an unexpected circumstance. In the circumstance of an emergency, the participants believed that transferring to hospital became the new best and safest. One participant had prepared for:'I felt empowered to be able to take a certain amount of control over my own care ... I think really largely for me, it’s really been a progressive experience of feeling more confident the more skilled up as time passed'. (FB06).Throughout the process of finding a better way, the participants simultaneously became the experts. They came to value their own ability to make informed and safe choices over that of their care providers. One woman described:'It was my third birth. I think that is a big factor. If it was my first birth, I probably would have listened to the obstetrician and just gone for the elective caesarean out of fear, so again I was very comfortable with birthing babies by this stage, very confident in my own ability'. (HB06).Another woman spoke of how her previous birth experiences had turned her into an expert. Having gained confidence and expertise through her previous birth experiences, she felt confident to birth her breech baby at home:birth outside the system.The participants described themselves as different to other women because they are ‘always bucking against the system’, ‘take responsibility’, ‘investigate to ensure that they know’, ‘believe in their ability to ‘know,” ‘have a sense of entitlement to choose’, and ‘possess confidence in their ability to birth’. These characteristics facilitated the development of their expertise as they pursued a better way and ultimately led them to birth outside the system. In this study Birth outside the system was considered to be the best and safest option compared to other birthing options available. These findings bring into question the authority of biomedical ways of knowing and managing birth.This Grounded Theory study aimed to explore what motivates women to Women who choose to give birth outside the system hold the belief that they, rather than the socially defined birth experts, hold the authority to define risk and safety. Authoritative knowledge describes the knowledge that ‘counts’ in a particular situation, is the basis on which decisions can be made, and provides justification for a course of action . AuthoriWomen seek to give birth where they feel safe and make decisions that prioritise the health and well-being of their baby . HoweverThe rhetoric of biomedical birth practices argues that interventions are employed in order to enhance safety , 44. HowCurrent risk discourse dictates that giving birth is risky and hospitals are the safest place to give birth. However, women who gave birth outside the system believed that, although childbirth carries an element of risk, that a hospital is not the safest place, or an effective way to mitigate risk. Bisits (2016) suggests that the risks associated with pregnancy and birth are low prevalence phenomena, however there is an obstetric tendency towards an emphasis on risk. He suggests that when presented with the range of ways pregnancy and birth can go wrong, women begin to feel that pregnancy is dangerous . PerpetuIt was apparent in this study that women considered risk seriously but placed the iatrogenic risks of giving birth in a hospital under intense scrutiny, challenging implicitly agreed assumptions that hospital birth must be safer and exposing risks that are often simply accepted as part of birth . In thisbirth outside the system came from a desire to prevent a repeat of past traumatic events.Globally, bewteen 20–48% of women are reporting their birth expereince as traumatic . SimilarThe body of literature about the role of care providers in birth trauma is growing and shines the spotlight on maternity care providers as the instigators of events that have left women feeling traumatised , 53. It The difference between a biomedical and humanised approach to maternity care is whether or not the woman giving birth retains control . The strbirth outside the system in order to preserve their autonomy. Similarly, Wagner (2001) suggests that if a woman wishes to control and humanise her birth, she must move outside of the hospital [The women in this study demonstrated that their concept of safety included ‘being the authority at my birth,’ which meant being able to control their birthing circumstances. The feeling of being in control is a significant factor in women perceiving their birth experience as satisfying and positive . A commohospital .birth outside the system and experienced poor perinatal outcomes were not included in this study. As all the participants had a live and overall healthy baby, this study is unable to provide insight into the thoughts of women who experienced adverse outcomes and how they feel about their birth choice after the fact, though other researchers have begun addressing this [The limitations of this research include that it was undertaken in Australia so may not be generalisable to other countries. Aboriginal and Torres Strait Islander women were not interviewed for this study and are a group known to avoid mainstream services in Australia. In addition, the study group was not culturally diverse, with the majority being white Caucasian, socially advantaged women. Women who chose to ing this  .Shortfalls in the Australian maternity care system were major contributing factors to women’s choice to give birth outside the system. If the Australian maternity system neglects to provide evidence-based care and limits birthing options, women will continue to birth outside the system. Improvements to maternity policies and practice should prioritise humanising maternity care by providing continuity of midwifery care models for all women including women with complex pregnancies, one-to-one midwifery care during birth, woman-centered care, and midwifery-led care in public homebirth programs and standalone birth centres. Finally, enhancing support for private midwifery services would significantly reduce the number of women opting for freebirth as, based on this research, women would be more likely to access a midwife in order to birth at home over choosing freebirth. Perhaps if women felt less disillusioned by their maternity care options and they felt safe within current maternity care options, they would not be forced to consider birth outside the system , 6. Adopbirth outside the system. In this study Birth outside the system was considered to be the best and safest option compared to other birthing options available. Shortfalls experienced within the Australian maternity care system were major contributing factors to women’s choice to birth outside the system.This Grounded Theory study aimed to explore what motivates women to


The last third of gestation is a period of high energy demand for the dam, because she needs to support the growth of fetuses and the newborn. Moreover, towards the end of gestation, maternal feed intake is reduced as the mass of the fetoplacental unit increases. The maternal diet often cannot meet nutritional requirements, compromising thermoregulatory capacity, wellbeing, and viability, and the survival of the newborn. We have shown that energy supplementation of the mother during the last third of gestation does not affect progeny birth weight but enhances mother–young bonding.n = 12); (ii) alfalfa + 150 g/head daily energy concentrate ; (iii) alfalfa + 300 g/head daily energy concentrate . At birth, we collected progeny data on birth weight, birth type, sex, rectal temperature, heart rate, respiratory rate, time to standing, time to udder connection, and time to first feeding. For the dams, we collected data on the duration of labor, time to clean the progeny, and time to allow first suckling. At birth, body weight, rectal temperature, heart rate, and the respiratory rate did not differ among treatments (p > 0.05). In the dams, labor duration was not affected by the treatments (p > 0.05). The T-150 dams were faster to clean the newborn and allow first suckling (p < 0.05). The T-150 progeny were faster to stand and the T-300 progeny were faster to connect to the udder (p < 0.05). We conclude that energy supplementation of the dam during the last third of gestation does not affect the birth weight of the progeny, but enhances the mother–young bonding.We tested whether maternal energy supplementation during the last third of gestation improves birth weight, neonatal wellbeing, and mother–young bonding. Thirty-six pregnant French Alpine goats were randomly allocated among three nutritional treatments for the last third of pregnancy: (i) Control, fed alfalfa (T-0;  Goats are distributed across diverse geographical and agro-ecological zones around the world, with nearly 95% of them found in developing countries . ImportaWith respect to nutrition, the last third of gestation and early lactation are of interest because they are periods of high maternal energy demand to support the growth of fetuses and the newborn ,7. ArounFrom birth, the newborn quickly adapts to the extra-uterine environment by modifying its homeothermy mechanisms—core body temperature, heart rate, and respiration rate ,25. DespConsidering this situation, it is clearly necessary to develop simple, low-cost strategies that will allow goat farmers to increase the productivity and reproductive efficiency of their animals. Such strategies will precisely coordinate nutritional inputs with the stages of the reproductive process to ensure that the metabolic signals are appropriate for optimizing the wellbeing and vitality of the neonate. Energy supplementation two weeks before parturition has been reported to increase colostrum quality and neonatal survival ,37, but This study was conducted during the breeding period at the Caprino Genetic Improvement Center in northern Mexico . All procedures were consistent with the International  and NatiFrom 55 multiparous French Alpine goats that had been naturally mated with experienced bucks for 42 days (two full reproductive cycles), 36 females on the same day of pregnancy were selected and, along with their progeny , were used to investigate the effect of energy supplementation during the last third of gestation (from day 100 of pregnancy to delivery) on birth weight, neonatal wellbeing, and mother–young bonding. All goats were managed, including deworming and vitamin supplements, according to the standard procedures of the Genetic Improvement Center, and they had free access to clean water and a block of mineral salts containing at least 17% P, 3% Mg, 5% Ca, and 75% NaCl.Pregnancy, number of fetuses, and gestational age were assessed three times, between 30 and 45 days after the start of mating, by transabdominal ultrasonography . Gestational age was estimated by assessing fetometric parameters: uterine depth (in early pregnancy), fetal crown–rump length, fetal biparietal diameter, and calcification of the fetal ribs and skull ,41,42.2 pens, one for each dietary treatment, ensuring the average body weights of the groups were similar. The treatments were T-0 ; T-150  and T-300 . The nutritional content of the diet and the energy concentrate is reported in On estimated gestational day 100 ± 4, the goats were randomly allocated among three 120 mOn the day of kidding, date, sex, birth type, and birth weight were recorded. An hour and a half after the end of the kidding process, the kid rectal temperature was determined , and the respiration and heart rates were estimated with a stethoscope . A total of 66 kids were born, of which 3 died in the days after birth (2 from T-0 and 1 from T-150); their data were not included in the analysis.Maternal and neonatal behaviors were observed by an experienced operator standing approximately 10 m away from the new mother and its progeny. This approach was backed up with a closed-circuit system in which 6 cameras were placed on the top and side of the pens, providing a panoramic view of each pen . The onset of parturition was detected when the behavior of the dam began to change, with the goat moving away from the others, becoming restless , and vocalizing. The time of the first contractions was noted as the start of labor. The following variables were then recorded for the dams: labor duration, time to clean the newborn, and delay to first suckling. The following variables were recorded for the newborn: delay from birth to standing and delay to first connection to the udder.p = 0.2328). Subsequently, a completely randomized design using linear mixed model procedures (PROC-MIXED) in SAS [A Shapiro–Wilks normality test was used to show that the data were normally distributed  in SAS  was usedp > 0.05) interactions were removed from the model. Finally, an analysis of orthogonal polynomials was carried out to compare treatments.All 2-way interactions among fixed effects and covariates were included in each model, and non-significant  to be heavier (3.8 ± 0.8 kg) at birth than twins (3.7 ± 0.5 kg) or triplets (3.3 ± 0.7 kg). The difference did not differ statistically , the average heart rate was 188 bpm (range 136–240 bpm), and the average respiration rate was 74 bpm (range 36–180 bpm), and treatment did not affect these measures of neonatal wellbeing  and did not differ among treatments , with T-150 kids being faster and T-300 kids being slower compared to control kids (Contrary to expectations, energy supplementation of multiparous female goats during the last third of gestation did not affect the birth weight of the progeny, their rectal temperature, heart rate, or respiration rate. On the other hand, mother–young bonding was improved with the T-150 supplement, as evidenced by the more rapid cleaning of the newborn and the shorter delay to standing. With respect to the delay in the first attachment to the udder, the situation was not so clear, with T-300 kids being faster and T-150 kids being slower. In general, these observations are consistent with the expected effects of maternal energy status on mother–young bonding behaviors, the thermoregulatory capacity of the newborn, and neonatal wellbeing.The maternal diet offered during the last third of gestation did not meet energy requirements, yet the birth weight of the progeny was not significantly affected by treatment, extending on observations by Mellado et al.  in crossMaternal energy supplementation during the last third of gestation did not affect the rectal temperature of the neonates. A higher rectal temperature indicates a greater thermoregulatory ability to adapt to the extra-uterine environment and is associated with increased viability and survival . InteresGlucose circulating in the mother and fetus would probably have been increased by the energy supplements fed during the last third of gestation, leading to an improvement in the physiological status of the neonate , but we Many previous reports have shown that, at birth, males are heavier than females and singletons are heavier than twins or triplets ,21,22,42The duration of labor increases as the weight of the progeny increases because large fetuses cause dystocia , affectiThe ability of the neonate to stand and find the udder quickly may be related to an increase in the circulating concentrations of cortisol and glucose , leadingIn conclusion, maternal energy supplementation during the last third of gestation in multiparous goats did not influence birth weight and had no consistent effects on measures of neonatal wellbeing. However, it improved mother–young bonding and would thus be expected to improve neonatal survival under field conditions. Further research is needed to elucidate the impact of gestational energy supplementation on post-weaning growth and the subsequent reproductive efficiency of the progeny.