12.1.1 Hematopoietic Stem Cell Transplantation

Over 10,000 bone marrow transplants are performed every year in the United States to treat patients with severe blood disorders [1]. Hematopoietic stem cells (HSCs), capable of giving rise to the entire hematopoietic system [2], are the key therapeutic component of the transplanted bone marrow units and are therefore widely used to treat many blood malignancies, including myeloma, leukemia, and lymphoma, as well as immune deficiencies and other hematological disorders [3–6]. In most cases, the patient’s own diseased hematopoietic system is first ablated and then reconstituted with healthy cells using HSC transplantation (HSCT). Following systemic transplantation, the first key cellular process that affects HSCT efficacy is the homing of HSCs to the bone marrow, where an optimal niche is provided for hematopoiesis [7]. Akin to leukocytes, HSCs roll on, adhere to, and migrate across the vascular endothelium at specific tissues, guided by chemotactic gradients [8,9]. Lack of homing capacity has been shown to drastically reduce HSCT efficacy [10]. After homing, engraftment is required to retain and maintain HSCs in a conductive microenvironment for long-term survival and proper function (e.g., self-renewal, differentiation). Enhancing homing and engraftment will likely improve efficacy and reduce the dosage of HSC infusion, allowing more patients to be treated with the same donor pool. This is important since HSCs are difficult to expand in vitro without inducing undesirable differentiation and cell death [11,12].

Because HSCT is typically allogeneic, a main challenge in HSCT is significant immune response following transplantation. Not only may the host immune response compromise the engraftment and survival of transplanted HSCs [13], patients are also at risk for graft-versus-host disease (GvHD), which can lead to death [14]. The risk of an immune response can be lessened via prophylactic immune suppression [15]. Haplotype matching based on the human leukocyte antigen (HLA) further ameliorates the risk, although this limits the number of potential donors [6]. Nowadays, HSCs can be efficiently harvested from multiple tissue sources, including cord blood, substantially increasing the number of patients who can benefit from HSCT [16,17]. Importantly, genetic modification enables the potential use of autologous HSCs: for some diseases, patients’ cells can be corrected ex vivo and returned to the same patient, providing a promising platform when matched donors are unavailable [18].

However, significant challenges still remain for HSCT. In addition to GvHD and poor engraftment of transplanted cells, disease relapse often occurs after allogeneic (unmatched), haploidentical (matched), or autologous transplantations [19]. Although continuous efforts are made to engineer cells to improve therapy, successful implementation into the clinic is challenging due to safety issues and preservation of engineered properties post-transplantation [20]. Genetic modification entails potential risks such as abnormal splicing, inactivation of tumor suppressor genes, or accidental oncogene activation, that may result in development of leukemia [21]. Nongenetic modifications may circumvent some risks while conferring better control over cell fate post-transplantation, as we will discuss in detail later.

12.1.2 Pancreatic Islet Transplantation

Another cell therapy in growing demand is pancreatic islet transplantation for treatment of type 1 diabetes (T1D) [22]. In T1D, the pancreas does not produce sufficient levels of insulin, mostly due to autoimmune destruction of beta cells, resulting in impaired glucose metabolism [23]. Patients with T1D need to constantly monitor their blood glucose and receive insulin injections to reduce hyperglycemia when necessary. Poor control of blood glucose may lead to hypoglycemia, which can be fatal, or chronic hyperglycemia, which may result in complications such as heart disease, kidney disease, and nerve damage [24,25]. Successful islet transplantation would restore autonomous, precise control of blood glucose, freeing patients from constant monitoring, and adjustment of blood glucose. Allogeneic islets are required for T1D [25], since the patient’s own islets are typically dysfunctional. Unfortunately, islets are composed of terminally differentiated cells and cannot be expanded ex vivo, while donor morbidity is clinically unjustifiable. Elaborate procedures for quickly obtaining fresh islets from cadavers are therefore required, especially given the fact that islets from at least two deceased donors are required to treat a single patient [26].

Like HSCT, allogeneic islet transplantation faces immune rejection and donor shortage. But unlike HSCs, islets are large multicellular aggregates containing multiple cell types and have to be administered locally instead of systemically (i.e., via the bloodstream) [27]. This greatly affects the repertoire of strategies for cell engineering. For example, genetic modification becomes more difficult since transfection efficiency will vary according to the location of the cell within the islet, whereas tissue engineered islet with defined compositional and structural features becomes instrumental for controlling cell fate and function following local transplantation [28]. However, while homing may be less relevant, engraftment and survival remain crucial for the success of islet transplantation: islets have to be vascularized quickly post-transplantation to maintain sufficient viability for long-term function [29].

Although the host immune response may be reduced by genetically downregulating foreign antigens [30–32], encapsulation inside biocompatible scaffolds such as alginate or porous pouches can sufficiently shield islets from both cellular and humoral components of the host immune system and thereby offer stronger and longer term protection [33–35]. Native islets deliver insulin directly into the hepatic portal vein; hence the gold standard has been intraportal implantation [36], which requires invasive surgery. Scaffold technologies may potentially enable minimally invasive [37] islet transplantation, such as subcutaneous implantation, given that a variety of problems will be addressed, such as capability of islets to detect changes in blood glucose, delivery of relevant amounts of insulin into the bloodstream, scaffold degradation over time, foreign body response against the scaffold, and nutrient transport to encapsulated islets [38]. Supplementing scaffold technologies with genetic strategies, such as accelerating vascularization by ectopically expressing pro-angiogenic factors [39], or promoting islet viability and functionality by ectopically expressing hormones [40] may be necessary to achieve sufficient efficacy of islet transplantation in the future [26]. An emerging paradigm is to derive pancreatic cells from precursors in vitro before transplantation [41,42]. Cells may then be administered systemically as single cells instead of aggregates, allowing them to efficiently home to and engraft at sites (e.g., hepatic or pancreatic capillary beds) where optimal islet physiology may be achieved.

12.1.3 Mesenchymal Stem Cell Therapy

Mesenchymal stromal cells, otherwise known as mesenchymal stem cells (MSCs), have emerged during the past two decades as promising candidates for cell therapy [43–45]. Unlike pancreatic islets, MSCs are readily isolated from bone marrow and other adult tissues without significant donor morbidity. Unlike HSCs, MSCs can be easily expanded ex vivo to obtain a sufficient quantity for cell transplantation [46]. Moreover, their immune-evasive and potent immunomodulatory properties following systemic infusion permit allogeneic transplantation and have prompted their use in over 380 clinical trials for treating multiple diseases, including lupus nephritis, GvHD, multiple sclerosis, and cardiovascular diseases [47]. While results from pre-clinical animal studies have been encouraging and hundreds of millions of allogeneic MSCs have been safely administered, clinical trials have produced only mixed results for efficacy endpoints and MSC translational potential has not yet been fully realized [48–51]. This could be attributed to the innate heterogeneity of MSC population, significant variability in their donor/tissue sources, manufacturing processes, as well as their diverse characterization methods [52]. One of the major challenges for MSC therapy is poor homing to diseased or damaged tissues, potentially due to minimal expression of key homing ligands on MSC surface [49,53,54]. Since the MSC secretome serves a crucial role in their therapeutic impact, increasing the number of transplanted MSCs at disease sites at the same total dosage will likely significantly improve the efficacy of MSC therapy [55].

12.1.4 Embryonic Stem Cells and Induced Pluripotent Stem Cells

Embryonic stem cells (ESCs) are perhaps the most promising yet most controversial cell type being investigated for therapy. Unlike adult stem cells, ESCs have an unlimited capacity for self-renewal, can differentiate into all three germ layers (ectoderm, endoderm, and mesoderm), and thus can generate almost any cell type [56]. However, adverse effects, such as formation of teratomas, and the many ethical issues that plague the use of ESCs, significantly hinder their translation into the clinic [57]. Numerous studies demonstrated the therapeutic potential of ESCs. For example, Min et al. [58] were able to significantly improve left ventricular function in post-infarcted rats after ESC transplantation. Brustle et al. [59] demonstrated that ESCs differentiate into oligodendrocyte and astrocyte precursors and induce axon myelination in a rat model of dysmyelinating Pelizaeus–Merzbacher disease. Besides cardiac and neural applications, ESC studies have spanned virtually every discipline of the biomedical field [60]. Better understanding of the mechanisms governing ESC differentiation and transformation may advance the development of safe and effective ESC therapies in the future.

Induced pluripotent stem cells (IPSCs) bypass ethical issues of ESCs and are also starting to be tested in clinical trials [61]. IPSCs are generated by reprogramming somatic cells to regain pluripotency [61,62], first demonstrated when Takahashi and Yamanaka [63] successfully reprogrammed adult fibroblasts into an embryonic-like state. Theoretically, this would enable a virtually limitless quantity of highly desirable ESC-like cells, while avoiding any ethical issues. One of the main questions is whether IPSCs are truly as pluripotent as ESCs [62]. Different groups have shown significant differences between IPSCs and ESCs. For example, the differentiation potential of IPSCs appears intrinsically lower than that of ESCs [64]. In addition, IPSCs seem to have an increased propensity to differentiate back into the cells they were formed from [65]. Despite these differences, IPSCs have been used in ample studies spanning a multitude of biomedical applications. For example, Wernig et al. [66] were able to demonstrate that neurons derived from IPSCs were able to functionally integrate into the brains of rats with Parkinson’s disease. Much of the potential of IPSCs remains untapped, however, making this field an exciting source of potential therapies.

Among the challenges discussed above, poor cell targeting to disease sites following transplantation seems to be a common drawback shared by all the aforementioned cell therapies. We will now discuss micro/nano-engineering approaches to control cell homing/targeting. These approaches mainly involve the cell surface, the key interface where the cell interacts with its environment during the homing process.

12.2.1 Affinity-Based Cell Targeting

Different bioengineering approaches have been exploited to modify the cell surface to enhance cell targeting to desired sites. A generalized concept is to trigger receptor–ligand interactions between the surface of transplanted cells and the cells at the target site. Cells can be engineered to overexpress biological or artificial receptors or antibodies to specifically target cells expressing the corresponding ligands or antigens [69] (Figure 12.1).

Whole and fragmented antibodies have been incorporated into cell surfaces to enhance cell localization to target sites. For instance, coating MSCs with whole antibodies against MAdCAM increased their homing to inflamed bowel and improved therapeutic efficacy on inflammatory bowel disease [70]. Likewise, in adoptive immunotherapy, autologous T cells may be modified with antibodies ex vivo and redirected to target specific cells, such as cancer cells. Whole antibodies have been engineered to create chimeric T-cell receptors (TCRs), which signal the T cell to launch a killing response upon binding to malignant cells [71,72].

Fragmented antibodies, such as single chain variable fragments (scFv) antibodies which do not contain the Fc domain but retain the high specificity and affinity of whole antibodies, have also been used to modify the cell surface [73]. Omitting the Fc domain eliminates potential binding of Fc receptors onto the cell surface and facilitates vector design for genetic engineering, but removes the signaling ability intrinsic to the Fc region. Fragmented antibodies have been engineered to create bispecific adaptors (diabodies) that link the specificity of two antibodies into a single agent and act as a bridge between two cell types. Cells have been genetically modified to secrete diabodies which bind tumor cells at one end and bind/activate T cells on the other, directing T-cell cytotoxic activity specifically toward tumors and significantly inhibiting tumor growth [74–76]. Fragmented antibodies, instead of whole antibodies, are preferred for this approach since they penetrate tissues better due to their reduced size [77].

These two approaches represent how both membrane-anchored and secreted factors may enhance cell targeting via the cell surface. The same concept extends to receptors beyond antibodies. We will next discuss how such factors may be imparted to cells that natively underexpress them.

12.2.2 Bioengineering Approaches to Accomplish Affinity-Based Cell Targeting

12.3.1 Nanosystem Types and Key Characteristics

The largest group of particulate carriers is NPs which encompass nanospheres, nanocapsules, micelles, dendrimers, nanocrystals, and nanogolds. NPs are chosen for their small size, high surface-to-volume ratio, and surface functionality. The most commonly used NP materials are polymers and lipids, which typically encapsulate hydrophobic drugs. Polymer-based NPs are made from synthetic polymers such as poly(lactic acid) (PLA) and poly(lactide-co-glycolide) (PLGA) or natural polymers such as gelatin, alginate, collagen, and chitosan [124–126]. PLA and PLGA NPs are especially attractive since their degradation time can be tuned from days to months based on copolymer configuration and molecular weight [127]. However, without special treatments to render their surfaces hydrophilic, they are unstable in the aqueous biological environment and are unsuitable for systemic delivery. Lipid-based NPs typically have an amphiphilic coating to stabilize their hydrophobic cores in an aqueous environment. There are two common types of lipid-based NPs: solid lipid NPs [128] which consist of a solid lipid core and lipid nanocapsules [129] which have an oily (liquid) center [130]. Although lipid-based NPs may be more biologically stable, tuning their biodegradation in vivo is challenging. Other lipid-based NPs include micelles and liposomes, with liposomes being particularly attractive for their ability to encapsulate hydrophilic molecules [131].

Nucleic acids represent an important class of therapeutic hydrophilic molecules. Safety concerns over viral vectors have shifted research focus toward nonviral vectors [132]. Cationic liposomes and cationic polymer-based vectors interact with negatively charged molecules such as DNA while maintaining an overall positive charge on the surface, enabling interaction with the negatively charged cell surface and subsequent endocytosis, thus preventing DNA from systemic enzymatic degradation. Cargo is released into the cytoplasm via several mechanisms of endosomal escape such as membrane disruption/reassembly [133]. Similarly, cationized polysaccharides such as spermine-dextran [134] and spermine-pullulan [135] can be recognized by cell surface receptors to promote internalization, with biodegradability serving as another advantage. Although recent in vivo results have shown toxicity and low transfection efficiency, charged polysaccharides still demonstrated remarkable potential for use in tissue regeneration and cancer target therapy [91,136,137].

Another promising nanosystem is carbon nanotubes (CNTs) [138,139], which can immobilize and intracellularly deliver small molecules, nucleic acids, and proteins as long as the cargo can adsorb onto the CNT surface, typically by pi–pi stacking interactions [140,141]. Unfortunately, studies have also associated CNTs with cell toxicity [142], reducing its potential for clinical translation.

Protein adsorption on the surface of particles was originally viewed as undesirable as it promotes clearance by phagocytes. Efforts have since shifted toward using cellular binding and internalization properties to their advantage, such as to target specific cell types or to develop cellular drug carriers [143]. Internalization efficiency of particles by cells depends on cell type, particle size, surface chemistry, surface topography, shape, and mechanical properties [89]. Additional ligands can be conjugated to the particle surface to promote internalization via specific endocytic pathways [144].

12.3.2 Candidate Therapeutic Agents and Cell Types for Cell-Based Delivery

The types of therapeutic agents to be encapsulated in particles or cells depend not only on the disease of interest but also their stability during encapsulation and release kinetics after encapsulation. Encapsulation in cells warrants extra attention because the released drug may affect the cell and vice versa. For example, cell-based delivery of cytotoxic chemotherapeutics requires careful tuning of release kinetics, since excessive burst release may kill the cellular carrier before it reaches the target tissue [145]. An appealing strategy is to deliver prodrugs—drugs that are administered as inactive molecules, but are converted into their active form after specific chemical or enzymatic activity intracellularly or extracellularly. For instance, one can use the resident enzymes present in cells to convert prodrug into diffusible drug that can be released into the circulation [146]. Many therapeutic cargos, including nucleoside/nucleotide analogs [147], glucocorticoid analogs [148–151], enzymes [152–154], toxins [155,156], mRNA [98], peptides [157–167], and antisense peptide nucleic acids [168–171], are available as prodrugs.

An ideal cellular carrier must not only withstand its therapeutic cargo but also home to target tissues for efficient drug delivery. The most obvious, yet valuable, approach is to harness the innate homing properties of specific cells. Examples include leukocytes and stem cells that are known to home to tumors and sites of inflammation [172–175]. However, the innate homing properties may be suboptimal; hence cell surface engineering can be applied to maximize cell delivery to specific sites, as discussed in the previous section. The next section reviews a selection of cell types and their tropism. Discussion of cancer applications will be deferred to the following section, where we provide a case study on how multiple cell studies have been used as Trojan horses to target tumors.

12.3.3 Case Study: “Trojan Horse” Cell Therapy for Cancers

Tumors are characterized by chaotic vasculature, high interstitial pressure, and dense extracellular matrices, presenting major obstacles for treatment via systemically administered drugs or drug-loaded particles [126]. Often, drugs and particles are unable to penetrate into the tumor and, as a result, are rapidly cleared. An example is GBs, which are extremely malignant brain tumors, known for their rapid growth and vascularization [231]. Current treatments revolve around surgery, radiation, and chemotherapy with DNA alkylating agents such as temozolomide (TMZ). However, there are several limitations with current treatments such as the poor effectiveness of TMZ for TMZ-sensitive GB patients as well as GB resistance against chemotherapy and radiotherapy. In addition, glioblastoma stem cells (GSCs) possess infiltrative, proliferative, and progressive characteristics, rendering them more resistant to hypoxic and acidotic tumor microenvironments [232]. Although GBs are highly vascularized, penetrating the BBB is yet another challenge toward effectively delivering therapeutic agents to the tumor. In fact, the BBB prevents uptake of large molecules and of over 98% of small-molecule drugs available [233]. Likewise, NP transport efficiency across the BBB is poor. To deliver antitumor agents across the BBB and deep into GBs, cellular carriers—namely monocytes and stem cells—are attractive candidates due to their ability to infiltrate into gliomas [234–238].

Known for their tropism toward intracranial tumors, NSCs have been used to transport toxic molecules [239–244], proliferation inhibitors [245], anti-angiogenic agents [246,247], and cytokines with toxic molecules [240]. Additionally, NSCs have been genetically modified to yield cytokines such as IL-18, IL-2 [248–250], IFN-γ [251], and tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) [252–254] for modifying increased immune response at gliomas. NSCs have also been engineered to secrete cytosine deaminase (CD) that activates the prodrug 5-fluorocytosine (5-FC), so that cytotoxic effects of the systemically administered drug are localized within the tumor [189]. Off-target homing of antitumor NSCs is potentially dangerous as antitumor agents can harm healthy cells. To reduce off-target actions, overexpression of a pH-sensitive viral fusogen on the NSC surface has been used to fuse NSCs specifically with tumor cells, since tissue pH is lower at tumor sites [255]. These engineered NSCs carried a suicide gene, allowing selective killing of any syncytium formed by the NSCs and residual, unfused NSCs.

Monocytes or macrophages can also cross the BBB, but unlike NSCs, monocytes are isolated with much less invasive procedures. Choi et al. envisioned loading monocytes with gold nanoshells such that they home to the tumor site, differentiate into TAMs, migrate and deliver the nanoshells into hypoxic regions of the tumor. Once in place, near-infrared illumination would be used to heat the nanoshells and destroy the TAMs and adjacent tumor cells [205]. Nanoshell-based photothermal ablation therapy has shown promising results toward reducing tumor in mice, with over 90% remission rate [256]. Combining these concepts, monocyte carriers of gold nanoshells were successfully used to ablate human breast carcinoma tumor spheroids [172,205,219]. In another study employing a murine glioma model, cyclodextrin-based NPs were internalized by TAMs. Due to their tumor-tropic properties, the TAMs migrated within and around the tumor, demonstrating their capability to distribute therapeutic agents across the tumor [257]. T cells have also been used for nanoshell-based photothermal ablation [258], demonstrating the general effectiveness of leukocytes as tumor-homing cellular carriers.

Another use of T cells as a cellular vehicle followed from observations that viral particles can “hitchhike” on the T-cell surface, be taken up by tumor cells in vivo, and be released at the tumor site [259]. Extending the success of this strategy, numerous groups have used tumor antigen-specific T cells to carry oncolytic viruses to tumor deposits [260–262]. The same strategy can be applied to deliver NPs for cancer diagnostics and therapeutics, where the use of viruses is undesirable. Such agents include small-molecule drugs, antibody–drug conjugates, aptamers, and magnetic imaging agents [263,264]. The Irvine group used thio-reactive maleimide groups to crosslink nanocarriers, surface proteins, or lipids to the T-cell surface for up to a week. Their in vivo results showed successful biodistribution of T-cell-coupled NPs in tumor models, while systemic injection without cellular carriers resulted in fast clearance of the particles in liver and spleen [265].

MSCs are yet another cell type that exhibits tumor-tropic properties. Systemically administered MSCs carrying intracellular PLA and lipid nanocapsules demonstrated successful delivery of nanocapsules to gliomas [175]. NPs have also been conjugated onto the MSC surface using biotin–streptavidin bridges without attenuating MSC tropism toward tumor spheroids [266]. In multiple studies, MSCs were genetically engineered using viral and nonviral vectors to secrete immunostimulators such as IL-2, IL-12, IFN-beta, IL-18, and IL-7 to suppress tumor growth in glioma and other cancer models [203,250,251,267–276]. MSCs are also being used to deliver oncolytic adenoviruses to tumor sites [277,278]. More complex systems combining gene modification and prodrugs such as the CD/5-FC system (also applied on NSCs) have been tested on MSCs [279,280]. Another system uses herpes simplex virus thymidine kinase (HSVtk), which activates the chemotherapeutic prodrug ganciclovir [281]. Retrovirally transfected MSCs carried HSVtk to the tumor site, such that systemically administered ganciclovir was preferentially activated in the tumor, resulting in tumor suppression in GB patients [281]. The same system appeared to be less efficacious when NSCs are used instead [241,282–284]. Yet another combination involves rabbit carboxylesterase and CPT-11, in which genetically modified adipose-derived MSCs were used to deliver carboxylesterase that enzymatically cleaves the CPT-11 prodrug to release the active drug SN-38 to combat brainstem gliomas [285]. Despite these advances, thorough investigation is required to fully elucidate the interaction between MSCs and tumor environment, especially when MSCs have been suggested to promote tumor growth [286], and clinical trials have not shown long-term engraftment of MSCs in tumors [287].

While most cellular drug delivery vehicles are still in early stages of research, one cell type has been successfully translated into FDA-approved therapy. DCs have become a part of FDA-approved therapeutic cancer vaccine (against prostate cancer), in which antigen-loaded autologous DCs are injected into patients [288]. In addition, another group has proposed a method to track cells that have been loaded with NPs. Noh et al. encapsulated near-infrared fluorophores and iron oxide NPs together with model antigens such that cells carrying the NPs could be tracked in real time. Injection of those DCs resulted in tumor suppression [289]. DCs also show tropism toward lymph nodes which enables the use of DCs to carry immunosuppressive drugs to lymphoid tissues in order to suppress T-cell proliferation [290].

Apart from solid tumors, blood cancers may also be treated with cellular carrier. In a pilot clinical trial, autologous RBCs were loaded with asparaginase so they could remove asparagine—a nonessential amino acid—from blood. Asparagine has been shown to increase lymphoblastic proliferation which is crucial for the progression of lymphoblastic leukemia [291].