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Discov Nano
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10.1186/s11671-024-04105-6
Brief Communication
Delivery of PAMAM dendrimers and dendriplexes across natural barriers (blood–brain barrier and placental barrier) in healthy pregnant mice
Kuhn Eric 123
Srinageshwar Bhairavi 123
Story Darren T. 2345
Swanson Douglas 6
Sharma Ajit 6
Dunbar Gary L. 1234
Rossignol Julien rossi1j@cmich.edu

123
1 https://ror.org/02xawj266 grid.253856.f 0000 0001 2113 4110 College of Medicine, Central Michigan University, Mount Pleasant, MI 48859 USA
2 grid.253856.f 0000 0001 2113 4110 Program in Neuroscience, Central Michigan University, Mount Pleasant, MI 48859 USA
3 grid.253856.f 0000 0001 2113 4110 Field Neurosciences Institute Laboratory for Restorative Neurology, Central Michigan University, Mount Pleasant, MI 48859 USA
4 https://ror.org/02xawj266 grid.253856.f 0000 0001 2113 4110 Department of Psychology, Central Michigan University, Mount Pleasant, MI 48859 USA
5 grid.262914.a 0000 0001 2178 1836 Department of Psychology, Saginaw Valley State University, University Center, MI 48710 USA
6 https://ror.org/02xawj266 grid.253856.f 0000 0001 2113 4110 Department of Chemistry and Biochemistry, Central Michigan University, Mount Pleasant, MI 48859 USA
12 9 2024
12 9 2024
12 2024
19 1 14810 6 2024
6 9 2024
© The Author(s) 2024
2024
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Gene therapy is an important tool for treating fetal diseases that allows for the delivery and integration of therapeutic genes into the genome of cells carrying mutations. Nanomolecules, like PAMAM dendrimers, have recently come into wider use for carrying vectors as they have several advantages over viral vectors. Namely, (1) tunable size and surface chemistry, (2) uniform size, (3) the ability to target specific tissues, and (4) the ability to carry large biomolecules and drugs. Recently, we demonstrated that 4th generation (G4) PAMAM dendrimer with a cystamine core and a non-toxic surface having 90% –OH and 10% –NH2 groups (D-Cys) could cross the blood–brain barrier following injection into the bloodstream. In the current study, as a proof of concept, we delivered the dendrimers alone (D-Cys) tagged with Cy5.5 (D-Cys-cy5.5) to healthy pregnant C57BL/6J mice to determine the fate of these dendrimers in the pregnant mice as well as in the fetus. Systematic diffusion of the D-Cys-cy5.5 was evaluated on gestational day 17 (3 days after injection) using in vivo imaging. This revealed that the dendrimer was taken up into circulation and away from the injection site. Analysis of sections by fluorescence microscopy showed that D-Cys-cy5.5 was able to successfully cross the maternal blood–brain barrier. However, analysis of the fetal brains failed to detect dendrimers in the central nervous system (CNS). Instead, they appeared to be retained in the placenta. This is one of the first studies to analyze the distribution of surface-modified PAMAM dendrimer in the pregnant mouse and fetus following systemic injection.

Supplementary Information

The online version contains supplementary material available at 10.1186/s11671-024-04105-6.

Keywords

PAMAM dendrimers
Pregnancy
Placental barrier
Blood brain barrier
Fetal medicine
http://dx.doi.org/10.13039/100000002 National Institutes of Health 1R21EY030012-01 issue-copyright-statement© Springer Science+Business Media, LLC, part of Springer Nature 2024
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pmcIntroduction

Nanotechnology, particularly dendrimers, represents a new and potentially effective modality for the delivery of molecules throughout the body. Polyamidoamine (PAMAM) dendrimers are hydrophilic branching dendritic molecules that can range from 1 to 10 nm in diameter and are composed of three functional parts: (1) the core, which may be constituted by a cystamine, ethylenediamine, or diaminobutane linker, among others; (2) branching structures extending from the core; and (3) the surface functionalization. These nanomolecules, first created in the 1980s, are synthesized by one of two methods—either the convergent or divergent method [1]. The divergent method of synthesis polymerizes the molecules from the central core, building outward to the surface, while the convergent method begins with the surface functional groups and proceeds inward to attach the groups to the arms of the core. Certain features of the dendrimer, such as diameter, number of surface groups, and density, depend on the generation of the molecule, which refers to the number of layers of branching that are present. G0 (generation 0) dendrimers are 1 nm in diameter and have four surface functional groups, G1 dendrimers are 2 nm in diameter with 8 surface functional groups, and so on. The utility of dendrimers partly stems from their ability to be chemically tailored for diverse applications allowing optimization of solubility, as well as absorption, distribution, and excretion characteristics. They additionally have unique benefits, such as easy modification of their functionality, size, and charge through organic reactions. They have the ability to either attach cargo to their surface or to encapsulate the cargo within their branching structure. The cargo could be chemotherapeutic compounds like methotrexate, small interfering RNA [2], peptides, or antibiotics to target specific organs or tumors. Surface moieties may also be modified by the addition of fluorescent dyes or other molecules for tracking purposes. Dendrimers can be administered orally, transdermally, and parenterally [3]. Demonstrated medical applications of dendrimers have included topical delivery of steroids to the ocular compartment [4], targeted delivery of enoxaparin to the lungs [5], and treatment of neurological pathologies such as glioblastoma [6], among many other uses [7–9]. The cystamine core contains a disulfide bond (S–S) at the center of the dendrimer that can be reduced intracellularly (by glutathione or other reducing molecules), splitting the dendrimer into two dendrons and releasing any cargo within [2].

The ability to transfect cells with RNA and DNA to alter gene regulation and expression is essential to the development of gene based medical therapies. The use of CRISPR/Cas9 and small interfering RNA requires vectors both to protect the molecules from degradation as well as to facilitate uptake by target cells. Ideally, the vector would demonstrate stability within the plasma, be endocytosed into cells, and then release its contents. Traditionally, researchers have used viral vectors (such as adenovirus and adeno-associated virus). However, viral vectors can induce an immune response, and the stability of liposomes can be compromised by oxidation in vivo [10]. Various other delivery systems that do not utilize viruses have been developed, such as gold nanoparticles [11], liposomes, and zeolitic imidazole frameworks [12]. Dendrimers, however, demonstrate significant advantages in comparison in the packaging and delivery of nucleic acids due to their tunability and ability to protect against nuclease activity [13]. For example, small interfering RNA was successfully delivered to the brain by a carbosilane dendrimer in a mouse model to alter retrovirus replication [14]. PAMAM dendrimers, especially those at higher generations, have been most frequently used as carriers of plasmids with high transfection efficiency. Chahal and Khan were even able to use a low-generation PAMAM dendrimer to transfect lung epithelial cells with siRNA [15].

The distribution of therapeutics to certain anatomical sites, including the ocular compartment, the central nervous system (CNS), and the fetoplacental unit, is particularly difficult due to specialized physiologic boundaries such as the blood–brain barrier (BBB) and the placenta. These barriers are highly selective, with the protection offered by the blood–brain barrier mediated by tight endothelial cell junctions with pores of only 1–4 nm in diameter [16]. The BBB also has a thick basement membrane with astrocytic end-feet and fewer endocytic vesicles, limiting transcellular uptake. Thus, the BBB ensures tight control of the extracellular milieu to which neurons may be exposed. In a similar way, the placenta works to prevent toxins and pathogens from entering fetal circulation, although certain immunoglobulins and other molecules are able to cross via active transport and diffusion, depending on gestational age. The placenta separates the maternal and fetal circulatory systems by an endothelial cell layer as well as cytotrophoblasts and syncytiotrophoblasts [17]. While oxygen and carbon dioxide diffuse across this border easily, and other solutes may cross via active transport, it generally excludes structures larger than 1000 daltons [18]. Similarly, nanoparticle passage across the placenta is determined by size; however, composition also affects transplacental movement to a certain extent [19]. For example, gold nanoparticles had a size limit of 80 nm, while silicon nanoparticles [20], with much larger diameters, were able to cross [21].

While it has previously been shown that dendrimers are able to access highly protected brain tissue [22, 23], it is still unclear how well dendrimers containing cargo might be able to transit the placenta. The ability to selectively treat the mother without toxic effects on the fetus is an important consideration for many pathologies, including acute infections, as well as chronic diseases, such as diabetes and hypertension. Many medications, particularly antiepileptic drugs such as valproate, carbamazepine, and phenytoin, and antithyroid medications like methimazole, are associated with teratogenic effects (e.g., fetal hydantoin syndrome and aplasia cutis—[24]). Thus, these drugs are discontinued or changed during pregnancy. Furthermore, pregnancy can exacerbate diseases like Crohn’s and ulcerative colitis. Alternately, in some conditions, it is desirable to treat the fetus without an effect on the mother. Infections with TORCH pathogens, such as cytomegalovirus, toxoplasmosis, and zika, directly target the fetal brain. PAMAM dendrimers, with their versatility and control over distribution, offer a possible solution for these difficult medical situations [25].

The ability to effectively target the mother with drug therapies while minimizing fetal exposure is crucial. While intra-amniotic injection of dendrimers has resulted in uptake in fetal brains in a mouse model, there has not been demonstrated translocation of dendrimers across the placenta in vivo [26]. The goal of this study was to deliver our mixed surface dendrimers into healthy pregnant mice to assess their ability to cross the placenta and fetal BBB in vivo by characterizing their distribution in the pregnant mouse and the fetus. We used our established G4-90/10 (surface with 90% hydroxyl and 10% amine) PAMAM dendrimers for this study.

Materials and methods

PAMAM dendrimer synthesis

A G4-90/10 surface functionalized dendrimer utilizing a cystamine core was synthesized and tagged with the fluorescent marker cyanine 5.5 (D-Cys-cy5.5; Lumiprobe, Cockeysville, MD). Purification of the dendrimers was performed by overnight dialysis using a 0.9% sodium chloride solution, followed by dialysis in water. The dendrimers were subsequently lyophilized for about 48 h and stored at − 20 °C. Methods of characterizing the PAMAM dendrimers are detailed in our previous studies [22].

In vivo animals and groups

Nine pregnant female C57BL/6J (Jackson Laboratory, Bar Harbor, ME, USA) mice bred in-house between 5 and 15 weeks of age were used in this study. Mice were housed in transparent polycarbonate containers with wood chip bedding with ad libitum access to food and water. The male mates were removed after pregnancy was noted. The room temperature was kept at 22 °C, and room lighting was on a 12 h light/ dark cycle (lights ON at 12 AM). All procedures followed the guidelines of the IACUC (Aug 16, 2018, registered under the CMU IACUC protocol #18-23). The animals were divided into two groups: D-Cys-cy5.5 (n = 6) and Hank’s balanced salt solution (HBSS) group (n = 3).

Dendrimer dosing

On embryonic day 14, all animals received 150 μL of either HBSS or D-Cys-cy5.5 (20 mg/mL) via a single intraperitoneal injection. Pregnant mice were scruffed, and a 31-gauge needle was inserted, with the bevel facing upward, into the lower right quadrant of the abdomen towards the head at a 30°–40° angle to horizontal, and the plunger was withdrawn slightly to verify correct placement and assess for perforation. The plunger was then depressed slowly, expelling the entire volume of the injectable.

In vivo imaging using IVIS

In vivo imaging was performed on gestational day 17 (3 days after injection). Fluorescent imaging of animals was obtained with the IVIS Lumina LT In vivo Imaging System using Living Image 4.5.2 software (RRID:SCR 014247) in both supine and prone positions. IVIS is an optical imaging device that allows for non-invasive longitudinal monitoring of disease progression, fluorescent tracking, and gene expression patterns in live animals. Prior to imaging in the IVIS, the animals were anesthetized with 2% isoflurane gas and 0.8% oxygen. The fluorescent signal from D-Cys-cy5.5 was detected at an emission wavelength of 675 nm.

Tissue processing and histology

On the last day of gestation (day 21), pregnant mice were sacrificed via cervical dislocation, and the fetuses and placentae were extracted. Following extraction, the placentae, fetal brains (5 to 11 fetuses per pregnancy), and maternal brains were immersed in 4% PFA for 24 h at 4 °C, then transferred to gradually increasing sucrose solutions of 10%, 20%, and 30% for 24 h each at 4 °C. The brains were then frozen using 2-methylbutane (Sigma Aldrich, St. Louis, MO, USA) and stored at − 80 °C until used for sectioning. The brains were sectioned in the sagittal plane at 30 μm of thickness using a cryostat. The tissue was stained using Hoechst 33342 (Thermo Fisher Scientific) at a concentration of 0.01 mg/mL. The sliced tissue was subsequently mounted on positively charged microscope slides (Globe scientific Inc, Paramus, USA). Images were collected with a Zeiss Axio Imager M1 microscope (Carl Zeiss AG).

Results

Presence of D-Cys-cy5.5 in the maternal abdomen following IP injections

IVIS fluorescent imaging showed the presence of the D-Cys-cy5.5 in the abdominal area of the pregnant mouse (A) compared to the control mouse (B) when placed in the supine position (Fig. 1). No fluorescent signal was seen with mice in the prone position (data not shown). See the supplement figure for IVIS images from all mice.Fig. 1 Presence of the PAMAM dendrimers D-Cys-cy5.5 in the abdominal area of the pregnant mouse following intraperitoneal injections (A) compared to the control mouse (B)

Presence of PAMAM dendrimers in the maternal brain

The D-Cys-cy5.5 was distributed throughout the maternal brain following IP injection into the pregnant mothers at E14. The D-Cys-cy5.5 was taken up by all the cells in the brain as evidenced by the co-localization between the Hoechst and D-Cys-cy5.5 (Fig. 2).Fig. 2 Uptake of D-Cys-cy5.5 by brain cells in the maternal brain following intraperitoneal injections. Figure A shows the nuclei of the brain cells stained with Hoechst; B shows the fluorescence from the D-Cys-cy5.5 PAMAM dendrimers and C shows the merger between Hoechst and D-Cys-cy5.5 confirming that the D-Cys-cy5.5 has been taken up by the brain cells. Scale bar = 100 microns

Presence of PAMAM dendrimers in the placenta but not in the fetal brain

Upon imaging the placenta, we found that the D-Cys-cy5.5 was co-localized with the placental barrier, as shown in Fig. 3. Analysis of the fetal brains did not demonstrate the presence of the D-Cys-cy5.5, as shown in Fig. 4, confirming that the placental barrier prevented penetration of the PAMAM dendrimers to the fetus.Fig. 3 Uptake of D-Cys-cy5.5 by the placenta following intraperitoneal injections of the dendrimers. Figure A shows the nuclei of the placental cells stained with Hoechst; B shows the fluorescence from the D-Cys-cy5.5 PAMAM dendrimers; and C shows the merge between Hoechst and D-Cys-cy5.5 confirming that the D-Cys-cy5.5 has been taken up by the placental barrier. Scale bar = 100 microns

Fig. 4 No uptake of D-Cys-cy5.5 by the pup brain following intraperitoneal injections of the dendrimers. Figure A shows the nuclei of the brain cells stained with Hoechst; B shows no fluorescence from the D-Cys-cy5.5; and C shows the merge between (B) and (C) confirming that the D-Cys-cy5.5 has not been taken up by pup brain. Scale bar = 100 microns

Discussion

During pregnancy and following birth, many studies have observed changes in the maternal brain, especially functional changes related to memory and cognition [27]. Transcriptome analysis has revealed many changes in the gene and protein expression leading to the observed changes in the maternal brain [28]. Further, studies have elucidated the crossing of virus and other pathogen from the maternal circulation to the fetus causing fetal brain disorders such as autism schizophrenia and microencephaly as observed during Zika virus outbreak. There are multiple drugs and treatments underway to treat fetal disorders and pregnancy complications [29]. Research has shown that nonpolar fat-soluble drugs can easily cross the placental barrier and reach the fetus. In addition, major ligand-receptors such as ephrin and integrin that are present in the placenta as well as in both fetal and maternal brain. Researchers are targeting them to develop therapeutics to cross the placental barrier and reach the fetal brain [30].

In this study, we demonstrated that surface-modified PAMAM dendrimers can cross the blood–brain barrier in pregnant mice following systemic injection during the gestational period and are taken up by the brain cells. We detected dendrimers in the placenta but not in the fetal brain tissue, suggesting that the dendrimers did not cross the placental barrier and reach the fetus. This is of great importance in the field of reproductive medicine in order to administer drugs to treat the pregnant mother without affecting the developing fetal brain.

Penetration, permeability of drugs and molecules across the BBB and placental barrier

Two major transportation molecules at the BBB are the ATP-binding cassette (ABC) transporter family (with 7 sub-families consisting of ion channels and receptors) and the solute carrier (SLC) family [31]. The ABC transporters protect the brain against xenobiotics but are not well equipped to differentiate between the flow of treatment drugs and toxicants. Though this is highly protective against the flow of toxic substances to the brain, the same mechanism, to some extent, hinders the penetration of treatment drugs. SLC transporters in the BBB help in the transportation of metabolites, sugar, neurotransmitters, and vitamins to the brain and eliminate the entry of toxic substances. One of the unique properties of the SLCs is that they are associated with cell-specific drug delivery and regulation since they are present on specific brain cells like neurons and astrocytes. SLCs function by passive transport of ions and molecules as opposed to ABCs, which function by active transport [31, 32]. Similar to the brain, SLC, and ABC family-based transportation facilitates drug penetration through the placental barrier. The size limit for drugs that can cross the placental barrier is about 1 KDa, however, ions are able to pass through the placental barrier by simple diffusion. There are multiple factors that play a role in determining the exchange of molecules between maternal circulation and the fetus. Some of these determinants include maternal protein binding, molecular weight, lipid solubility, and drug ionization [31, 33].

A normal blood–brain barrier pore size is about 1.4 nm to 1.8 nm, and the space between the tight junctions is about 20 nm. Glucose, amino acids, organic anions, fatty acids and cations, and nucleosides are some of the molecules that can readily pass through the BBB [34, 35] and the BBB allows passive diffusion of small, lipid-soluble molecules (< 400–600 Da) [36]. Permeability of the placental barrier is size-specific and depends on the molecular weight of the substances/drugs. Studies have shown that this barrier exhibits different permeability for molecules of varying sizes and that permeability changes throughout pregnancy, becoming more permeable as gestation progresses [37, 38].

Previous studies have shown that certain medications administered during pregnancy can lead to congenital defects or termination of pregnancy [39]. In some cases, the newborn child can have developmental delays and other long-term sequelae affecting them throughout their life. Therefore, the safety of medications prescribed during pregnancy is a critical factor for both the mother and the fetus [40]. This study suggests that dendrimers may provide a safe means of drug delivery during pregnancy, especially for treating diseases of the central nervous system. Epilepsy and seizures are the most common brain-related issues faced by pregnant mothers. Other neurological issues include cerebral venous thrombosis, ischemic stroke, and intracerebral hemorrhage [41, 42]. Thus, PAMAM dendrimers may be a safe vehicle to deliver drugs to the maternal brain during pregnancy without off-target effects on the developing fetal brain.

Limitation of the study

The authors acknowledge that the limitation of the work is having not performed IVIS on day 7 following injection (gestational day 21). This decision was consciously made as imaging on day 21 would interfere with the birth of the pups.

Supplementary Information

Additional file 1.

Acknowledgements

This research study was supported by the E. Malcom Field and Gary Leo Dunbar Endowed Chair in Neuroscience, the program in Neuroscience, the College of Medicine, and the John G. Kulhavi Professorship in Neuroscience at Central Michigan University.

Author contributions

EK: conception, design of the work, acquisition data, data analysis, interpretation of data, drafted the original manuscript. BS: conception, design of the work, data analysis, interpretation of data, drafted the original manuscript. DS: interpretation of data, revised the manuscript critically for important intellectual content. DS: revised the manuscript critically for important intellectual content. AS: interpretation of data revised the manuscript critically for important intellectual content. GLD: revised the manuscript critically for important intellectual content, funding, approved the version to be published. JR: conception, design of the work, revised the manuscript critically for important intellectual content, funding, approved the version to be published.

Funding

This research study was supported by the National Institute of Health (1R21EY030012-01), the E. Malcom Field and Gary Leo Dunbar Endowed Chair in Neuroscience, the program in Neuroscience, the College of Medicine, and the John G. Kulhavi Professorship in Neuroscience at Central Michigan University.

Data availability

Upon request to the corresponding author.

Code availability

Not applicable.

Declarations

Ethics approval and consent to participate

All procedures followed the guidelines of the Institutional Animal Care and Use Committee IACUC (Aug 16, 2018, registered under the CMU IACUC protocol #18-23).

Competing interests

The authors declare no competing interests.

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
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