STIMULI-RESPONSIVE BIOMATERIALS FOR DRUG DELIVERY TO IMPROVE CANCER IMMUNOTHERAPY AND CHRONIC WOUND HEALING

Sequential delivery of biomolecules is very important as many biologics underlying injury and disease follow an orderly and sequenced series of events. Here we developed and introduced for the first time a dual compartment biomaterial system with an outer compartment made of gelatin and inner compartment that is a ferrogel which can be magnetically stimulated in order to provide on-demand, sequential delivery of multiple bio-instructive payload. We studied the potential application of this dual-compartment biomaterial system in different therapeutic contexts that may benefit from on-demand sequential deliveries, such as in cancer immunotherapy and in chronic wound healing. Chronic wounds can be a result of arrest in the inflammation phase of healing. Although inflammation critically initiates repair and helps clear infections, a prolonged inflammatory reaction can cause considerable harm to the injury site. After an appropriate duration of inflammation, this inflammatory response can be shifted to a more pro-healing response through the delivery of cytokines like interleukin 4 (IL-4) and interleukin 10 (IL-10). These anti-inflammatory cytokines alter the phenotype of macrophages from pro-inflammatory (M1) to anti-inflammatory (M2), suggesting a potentially powerful drug delivery strategy if these cytokines can be delivered in a delayed manner. We hypothesize that the transition of macrophage phenotype from proinflammatory (M1) to anti-inflammatory (M2) can be controlled through sequenced delivery of interferon gamma (IFN-γ), followed by IL-4 and/or IL-10. The goal of this research was to develop a wound-healing hydrogel system that initially delivers proinflammatory IFN-γ, followed by magnetically triggered delivery of pro-healing (antiinflammatory) IL-4 and/or IL-10. Our biomaterial system was composed of twocompartments: (1) a porous gelatin outer compartment designed to recruit macrophages and establish an initial pro-inflammatory (M1) phenotype, and (2) a magnetically responsive alginate inner compartment which was designed to deliver IL-4 and/or IL-10 when magnetically triggered to shift the response to anti-inflammatory by promoting (M2) phenotype. We showed that we can have fast release of IFNg (Promotes M1 phenotype) and MCP-1 (recruits macrophages) initially from the outer compartment while holding on to IL4 and IL10 that is loaded in the ferrogel and have them burst release when applying the magnetic field. Biomaterial-based cancer immunotherapy strategies require materials capable of recruiting dendritic cells (DCs) and reprogramming them with cancer antigen and danger signal. This strategy requires the implantation of a biomaterial that is loaded with DC recruitment factors, danger signals, and cancer antigen. This co-delivery of danger signal and antigen results in DC activation and homing of cancer-antigen-presenting DCs to the lymph nodes, subsequently triggering an anti-tumor immune response from the host. However, danger signals and antigen diffuse out of the biomaterial while DCs are being actively recruited to the biomaterial. This may result in lower concentrations of these necessary reprogramming agents by the time DCs are recruited and consequently, lower quantities of activated DCs, and a reduced anti-tumor immune response. It is possible that sequential release of DC recruitment and reprogramming factors will enhance the number of reprogrammed DCs over simultaneous release, leading to improved anti-cancer immune responses. In order to test this, a material system with unique delivery capabilities must be developed. Therefore, we designed a biomaterial system capable of first recruiting DCs by initially releasing DC recruitment factors from outer compartment. This biomaterial can deliver reprogramming agents (i.e., cancer antigen) when magnetically stimulated only after a substantial population of DCs has been recruited. The results showed that we were able to deliver GM-CSF (DC recruitment factor) initially from the outer compartment followed by delivering HSP27 (model cancer antigen) when stimulated in the magnetic field

compartments: (1) a porous gelatin outer compartment designed to recruit macrophages and establish an initial pro-inflammatory (M1) phenotype, and (2) a magnetically responsive alginate inner compartment which was designed to deliver IL-4 and/or IL-10 when magnetically triggered to shift the response to anti-inflammatory by promoting (M2) phenotype. We showed that we can have fast release of IFNg (Promotes M1 phenotype) and MCP-1 (recruits macrophages) initially from the outer compartment while holding on to IL4 and IL10 that is loaded in the ferrogel and have them burst release when applying the magnetic field.
Biomaterial-based cancer immunotherapy strategies require materials capable of recruiting dendritic cells (DCs) and reprogramming them with cancer antigen and danger signal. This strategy requires the implantation of a biomaterial that is loaded with DC recruitment factors, danger signals, and cancer antigen. This co-delivery of danger signal and antigen results in DC activation and homing of cancer-antigen-presenting DCs to the lymph nodes, subsequently triggering an anti-tumor immune response from the host.
However, danger signals and antigen diffuse out of the biomaterial while DCs are being actively recruited to the biomaterial. This may result in lower concentrations of these necessary reprogramming agents by the time DCs are recruited and consequently, lower quantities of activated DCs, and a reduced anti-tumor immune response. It is possible that sequential release of DC recruitment and reprogramming factors will enhance the number of reprogrammed DCs over simultaneous release, leading to improved anti-cancer immune responses. In order to test this, a material system with unique delivery capabilities must be developed. Therefore, we designed a biomaterial system capable of first recruiting DCs by initially releasing DC recruitment factors from outer compartment. This biomaterial can deliver reprogramming agents (i.e., cancer antigen) when magnetically stimulated only after a substantial population of DCs has been recruited.
The results showed that we were able to deliver GM-CSF (DC recruitment factor) initially from the outer compartment followed by delivering HSP27 (model cancer       [16][17][18] and magnetic fields 19,20 . Magnetically responsive hydrogels (i.e., ferrogels) are particularly of interest to us because of their biocompatibility, response to benign magnetic signals, and potential flexibility in tuning the timing and rate of the deliveries. [21][22][23] Ferrogels can be made by mixing iron oxide particles in the gel matrix, allowing the gel structure to deform when exposed to magnetic fields. This deformation results in convective purge of the molecules contained in the gel's matrix. [24][25][26] Biphasic ferrogels were later developed with macroporous structures for improving the deformation capability of these gels while using a lower concentration of the iron oxide particles. This macroporosity improves biocompatibility of the gel with pore sizes that are significantly enlarged, enhancing molecular and cellular transport through the gel. 27 While ferrogels have been deployed to provide on-demand, magnetically triggered deliveries, regulation of complex biological processes requires the ability to deliver multiple bioactive compounds from the biomaterial (e.g., cytokines, proteins, etc., often in sequence). Therefore, we developed a dual compartment biomaterial system for delivering critical sequences of bioactive compounds in response to externally applied magnetic signals. This system is composed of an outer compartment in the shape of a hollow cylinder made with porous gelatin and inner compartment which is a cylindrical biphasic ferrogel nestled inside the hollow outer compartment. Each compartment of this system has different design requirements and release mechanisms. The outer 3 compartment is designed to diffusively release bioactive molecules that are capable of actively recruit a cell type of interest. The inner compartment is designed to harbor a bioactive molecule, only appreciably releasing it to the cells recruited to the outer compartment when instructed to do so in response to an externally applied magnetic gradient. Thus, at its most fundamental, this two-compartment biomaterial system provides a means to sequentially recruit and then modify the behavior of cells after implantation, with real-time control over the timing of this sequence. This ability to flexibly recruit and modify cells to and within a biomaterial structure is of great importance in far-reaching biomedical and clinical scenarios. However, this dissertation will focus on demonstrating sequential biomolecular release in a few contemporary applications: cancer immunotherapy and wound healing (and the associated regulation of the inflammatory response, tissue vascularization, and recruitment and differentiation of tissue specific cell types required for properly regenerating wounds).

Cancer immunotherapy
Cancer is a group of diseases, which is characterized by abnormal and out of  1A, moving from right to bottom). This causes the immature DCs to become mature and active (Fig 1A, bottom). Finally, these activated DCs are re-infused back in to the patient's body (Fig. 1A, moving from bottom to left). In the body, these activated and antigen-presenting DCs travel to the lymph nodes and present those cancer antigens to natural killer cells (T cells). This results in the body mounting an immunological attack against the cancer associated with that antigen.
While yielding some promising results (Fig 1, B, "cell based" survival curve in green, 70% survival in cancer-challenged mice after 90 days), there are some disadvantages to this ex vivo cell-based approach. One is that systemically administered vaccines have short signal duration. Cell-based vaccines they are also very costly and shown limited effectiveness 29 . Furthermore, the vast majority of DCs injected back in to the patient rapidly die and few activated DCs (estimated at only ~0.5-2%) are able to migrate to the lymph nodes which leads to the need for multiple administrations and higher doses. These multiple and higher doses cause systemic toxicity problems in the host 2 . Moreover, this method requires blood withdrawal, DC cell isolation, tumor biopsy, antigen extraction and processing, several DC modifications outside the body, and injecting of DCs back into the body. This, in turn, requires two patient procedures, high cost, and significant regulatory concerns 2 . Because of these disadvantages, indirect cancer immunotherapy vaccines have not been particularly successful into causing solid tumors to regress or increasing patient survival relative to standard treatments 3 .

5
In order to address some of these concerns, biomaterial-based cancer immunotherapy was introduced. In this strategy, an infection-mimicking material is introduced in vivo, providing a site to attract and activate DCs (Fig 1C,   the DCs. This results in even fewer DCs being activated and reporting back to the lymph node ( Fig. 1,C, iv and v), and therefore a sub-optimal anti-cancer immunogenic response may occur. Thus if a biomaterial system were capable of initially releasing DC recruitment factors and delaying the presentation of danger signals and antigen molecules, enhanced populations of activated DCs could be generated, resulting in stronger anti-cancer immunological responses. We will therefore aim to develop and test the reprogramming effectiveness of a biomaterial system capable of flexibly controlling the timing and rate of these molecular deliveries in response to externally applied magnetic fields. This biomaterial will result in a high degree of clinical significance by improving cancer survivability and immunity and may be applicable to a wide variety of cancer types. It will also provide a high degree of broad investigative significance by providing a system for examining how the timing and dose of recruitment and reprogramming agents impact immunological responses in general.
The hypothesis guiding this research is that DC activation can be improved by delaying the release of antigens until a vigorous population of DCs has been recruited to the biomaterial. Thus, the overarching aim for this research will be to develop an implantable biomaterial system capable of first releasing DC recruitment factors from its outer compartment, followed by magnetically triggered release of a model antigen. Such 8 a system may be capable of recruiting DCs and only delivering cancer antigen to recruited DCs at optimized times when stimulated by externally applied magnetic fields. This proposed biomaterial system consists of two compartments, similarly as described before ( Fig. 2A, i and ii). Compartment 1 will initially release DC recruitment factor (GM-CSF) and will have a functionality to maintain recruited DCs (by having a macro-porous structure ( Fig. 2A, iii)). This compartment is made from porous gelatin and is loaded with GM-CSF recruitment factor. Compartment 2 consists of a magnetically deformable porous alginate structure that only appreciably releases a model antigen (Heat shock protein 27 (HSP27)) when magnetically stimulated ( Fig. 2A, iv and v). This will result in the delivery of antigen in an on-demand, delayed manner. This combined system will be capable of sequentially releasing DC recruitment factors, followed by releasing antigen with magnetic stimuli (Fig.2B). The reason behind choosing these proteins for our work is as follows. The cytokine granulocyte-macrophage colony-stimulating factor (GM-CSF) has been identified as a stimulator of dendritic cell recruitment. 30 Heat shock protein 27 (HSP27) is used as cancer antigen and is often a target for cancer therapy drugs and the immune system. The massive release of HSP due to widespread tumor cell necrosis after cytotoxic drugs can lead to CD8+ T-cell-mediated anti-tumor immune responses. 31 However, for the purposes of this dissertation, HSP27 will be used as a model antigen. Optimizing the effectiveness of cancer antigen is beyond the scope of this work.

Chronic wound healing
The proposed two-compartment, magnetically responsive biomaterial system may also be of use in the field of chronic wound healing. In the United States, approximately 6.5 million patients are diagnosed with chronic wounds. The treatment of chronic wounds costs more than $25 billion annually and this is expected to grow due to the increasing cost of healthcare, an aging population, and a rise in the occurrence of diabetes and obesity worldwide. 32 The process of wound healing includes four major steps: hemostasis (blood clothing), inflammation, proliferation, and tissue maturation. 33 Even a slight perturbation in this process can disrupt proper healing, leading to chronic wounds.
Chronic wounds are often a result of arrest in the inflammation phase of healing. 33 Although inflammation critically initiates repair and helps clear infections, a prolonged inflammatory reaction can cause considerable harm to the injury site. After an We hypothesized that the two-compartment biomaterial system described above would be capable of (i) initially delivering macrophage recruitment factor and factors that would direct recruited macrophages to adopt a pro-inflammatory M1 phenotype and (ii) magnetically delaying the release of factors that would transition recruited M1 macrophages to anti-inflammatory, pro-healing M2 phenotypes. Specifically, we propose to design the biomaterial system to initially delivery IFN-γ and MCP-1 from the porous outer compartment and retain and magnetically release IL-4 and/or IL-10 from the inner ferrogel compartment. The goal of this research is to develop a wound-healing hydrogel system that allows for the investigation into how the duration of the inflammatory period impacts would healing. Namely, by altering the time at which magnetic stimulation is applied, the proposed biomaterial system will be able to regulate the time point at which the inflammatory response transitions into a pro-healing response. Critically, this is a simple parameter to alter between experiments, enabling rapid investigations into how the timing of these biological processes impact regeneration. Review of Literature

Introduction
The ability to produce temporally complex delivery profiles in an on-demand manner is pervasively needed in a wide range of biomedical and clinical scenarios, ranging from cancer treatment to tissue engineering. Stimuli-responsive biomaterials provide a potential means of providing on-demand regulation over temporally complex delivery profiles. 1-4 Some stimuli-responsive materials can be preprogrammed to respond to environmental cues such as pH 5,6 or temperature [7][8][9] whereas others can be triggered ondemand via externally applied signals. For example, materials can be engineered to release payloads in response to magnetic, electric, ultrasound, and optical signals. [10][11][12][13] The focus of this review is to outline and discuss the potential of magnetically responsive materials for providing on-demand regulation of complex therapeutic delivery profiles. This focus is founded in a number of key factors. First, magnetically responsive biomaterials typically contain magnetic particles [14][15][16] , which are widely used in biomedical applications. For example, magnetic nanoparticles 17,18 and magnetic liposomes 19 can be synthesized and used as drug carriers. The use of iron oxide nanoparticles as magnetic imaging contrast enhancers has made its way to clinical trials 20,21 and are extensively used in magnetic resonance imaging (MRI). 22 Magnetic 16 particles are also used for targeting purposes 23 and hyperthermia-based treatments (i.e., cancer treatment involving heating up the cancer cells to temperature between 43°C to 46°C when cell viability drops and becomes more vulnerable to chemotherapy and radiation). [24][25][26] Furthermore, magnetic fields similar to those used in these treatments are widely used in everyday scenarios (e.g., at airport security, at store exits, in MRIs, and even in some children toys) and have been demonstrated to be harmless to the human body. 22,27 Taken altogether, this well-established track record of using magnetic particles and magnetic field stimulation creates a strong precedence for their diagnostic and therapeutic use. This suggests that magnetic nanoparticle integration into drug delivery systems and remote stimulation using magnetic fields may be a viable option for achieving on-demand control over biomolecular deliveries.
This review will concentrate on hydrogel-based drug delivery systems that are integrated with magnetic particles, endowing them with on-demand delivery capabilities when subjected to remotely applied magnetic fields. Hydrogels are used significantly in drug delivery applications due to a number of desirable features. 28,29 For example, they can absorb water up to 30% of their dry weight and can be made from biocompatible polymers. They can also be chemically and mechanically modified to better interface with tissues. [30][31][32][33] Ferrogels are hydrogels that have been integrated with iron oxide particles in their polymeric network and their preparation method is very well established. [34][35][36] Here, we will categorize the drug release mechanisms from ferrogels and will discuss the tunable parameters effecting each release mechanism. This, in turn, may shed light on how magnetically responsive ferrogels can be designed to achieve magnetic control over more temporally complex drug delivery profiles. We will then proceed to highlight the importance of producing multi-drug release profiles in an on-demand manner and underscore the limited amount of work being done in this area.

2.2
Magnetically triggered release mechanisms

Inductive heating, agitation, or melting of polymer structures
Hydrogels can be designed to change phase (e.g., collapse, degrade, or swell) when inductively heated or agitated under magnetic stimulation, leading to release of payloads. [37][38][39] The super paramagnetic iron oxide nanoparticles (SPIONs) inside these hydrogels can absorb energy from high-frequency magnetic fields, which in turn leads to swelling or collapse of the hydrogel matrix due to changes in temperature. This inductive heating occurs when an alternating magnetic field (AMF) is applied to SPIONs. Heat is generated due to Neel and Brownian relaxation phenomena. 40,41 This energy then transfers to the hydrogel's polymer matrix which can lead to conformational changes such as polymer collapse or degradation ( Figure 1). 42 In a study by Hu et al., 43 the authors were able to show the pulsatile delivery of vitamin B12 loaded in the polymeric system.
This system was made by embedding iron oxide nanomagnets with average diameter of 40 nm within gelatin polymer network crosslinked with genipin. When a high frequency magnetic field was applied, the nanoparticles twisted and shaken, causing the polymeric network to decompose and release its content. Vibrations of the particles were also shown to increase the local temperature of hydrogel as well, which could potentially be problematic if this system were used for delivering more sensitive molecules such as proteins (which can denature at higher temperatures, rendering them bio-inactive). NIPAAm-based nanogels were incorporated in ethyl cellulose-based membrane made containing iron oxide nanoparticles. As in the previous study, this gel formulation was used due to its ability to change its volume at different temperatures When an alternating 19 magnetic field was applied to these nanogels, the iron oxide nanoparticles contained in the membrane heated up and rose beyond physiological temperatures to 50°C. This heating caused a ~400 nm decrease in the diameter of the nanogels which then led to an outward flux of sodium fluorescein from the drug reservoir ( Figure 2).

Physical changes in response to magnetic fields
Magnetic fields can be used to generate forces on magnetic particles and these forces can be used to directly impact physical structures in hydrogel that impact payload retention and release characteristics. For example, magnetic particles can be entrapped within a hydrogel's polymer matrix ( Figure 4, left). In the presence of a DC magnetic gradient (e.g., from a hand-held magnet), forces are exerted on the entrapped magnetic particles. This force moves the particles towards the magnet and hydrogel matrix deforms due to this particle movement (Figure 4, right). This deformation results in decrease in gel volume and convective purging of water and loaded drugs from the gel system

Tunable parameters affecting drug release
There are several material parameters that impact the retention and magnetically stimulated release of payloads from these biomaterial systems such as ferrogel pore size, magnetic particle concentration and size. As with other hydrogel-based materials, matrix porosity is a critical parameter influencing release characteristics. Generally, a more open more structure results in higher levels of release both unstimulated diffusive release and magnetically stimulated release. However, in many applications, it is desirable to achieve low-levels of unstimulated release and much greater levels of release upon magnetic stimulation. Thus, hydrogel porosity must be tuned to achieved desirably low levels of unstimulated release and desirably high levels of stimulated release. Hydrogel nanoporosity (i.e., the mesh structure of the polymeric matrix) can be tuned by altering polymer and crosslinker concentration. [31][32][33] Hydrogel macroporosity (i.e., large, often interconnected disruptions in the pores macrostructure) can drastically increase surface area of the gel as well as magnetic compressibility. 35 from ferrogels, sensitivity of PVA-based ferrogels to magnetic fields were studied in terms of permeability coefficient (P) and partition coefficient (H). Results showed that for optimum magnetization there is a critical parameter of free volume per nanoparticle that needs to be met. For their particular gel system, they found 17-34% iron oxide in PVA ferrogels was necessary for optimal magnetic sensitivity.
For both magnetic heating/agitation-and magnetic deformation-based delivery strategies, magnetic particle size also impacts release characteristics. For magnetic heating, the use of particles smaller than 20 nm reduces eddy currents which in turn restricts the heating of the particles. 23 Therefore, traditionally, SPIONs with diameters between 5-28 nm are used as they heat most efficiently when exposed to AMFs in the radio-frequency range (i.e., 100s of kHz), which are simple and relatively inexpensive to produce. 61 For magnetic deformation-based drug delivery strategies, magnetic particle size also plays a key role in determining delivery characteristics. For example, it was shown that when subjected to the same graded DC magnetic field, ferrogels made with smaller iron oxide particles (less than 50 nm particle size) had significantly lower deformation and drug delivery capabilities compared to ferrogels made with larger iron oxide particles (less than 5 µm in size). 35  Beyond material parameters, the parameters associated with the applied magnetic field itself play a key role in influencing magnetically stimulated release. Specifically, the amplitude, gradient, frequency, proximity, and directionality of the magnetic field can impact release profiles from ferrogels. Higher amplitudes can generate more magnetic heating/agitation and/or more force generation 38,62 , though when using larger, ferromagnetic particles the gradient of the magnetic field is more critical than the amplitude per se. Certainly, proximity of the magnetic source impacts both the amplitude and relative gradient of the magnetic signal, so placement of the ferrogel relative to the magnetic source is a critical consideration.
Regarding frequency, its impact on release can be quite complex. Finally, the orientation or directionality of the applied magnetic field can impact release characteristics. For example, hand held magnets can align or move magnetic particles in specified directions based on the field lines emanating from the magnet (which is, in turn, based on the way the direction that the magnetic is oriented/held). This orientation/alignment can lead to aggregation of the particles or compression of the gel matrix in specified directions. This can greatly impact release. For instance, when using the system described in Figure 6, 55 orientation of the magnet such that the magnetic PDMS disk (yellow on schematic) is pulled downward would result in tightening of the seal, restricting drug release. Only when pulled upwards would this system provide release. Likewise, for a biphasic ferrogel, 35 orientation of the magnet gradient must result in pulling the iron-oxide-laden region of the gel against the soft, deformable region to 29 appreciably trigger drug release. If the magnetic gradient is aligned otherwise, the ironoxide-laden region will not deform the deformable region, thus limiting drug delivery.

2.4
Perspectives on generating temporally complex drug delivery profiles using magnetically responsive hydrogels

Introduction
While the section above outlines the many number of parameters that can influence magnetically triggered release, this parametric abundance provides a wealth of strategies for customizing release properties and uncovers the potentiality of generating more complex delivery profiles using magnetic fields. The ability to generate more complex delivery profiles (e.g., temporally dynamic deliveries that change vs. time and delivery profiles that involve more than one drug such as sequential deliveries) is pervasively required in modern medicine. Illnesses and injuries are often associated with the interruption or distortion of natural sequences of biological events. Thus, therapeutically regulating these sequential biological processes requires sequentially delivering multiple bioactive therapeutics with the proper dosing, timing, and sequence. 29,67-70 Beyond sequential deliveries for therapeutic control over sequential biological processes, the temporal profile of single drug deliveries can be critical for optimizing therapeutic outcome. For example, chronotherapies involve pulsing the delivery of anticancer drugs to increase drug concentration when the tumor is metabolically active and to decrease it when not metabolically active. 29,67,71,72 These pulsatile delivery profiles can also help prevent the tumor from developing an adaptive 30 resistance to the anticancer drugs. 73,74 Thus, strategies must be developed to generate temporally complex delivery profiles (i.e., pulsed deliveries) and multi-drug, sequential delivery profiles. This section will explore existing strategies for obtaining these types of deliveries and propose potential strategies for obtaining these deliveries moving forward.

Existing strategies
A key advantage to using magnetically responsive hydrogels is that a magnetic field can be applied at times when changes in the delivery profile are desired. Simply put, changes in delivery profile can be regulated in an on-demand manner. Therefore, magnetically responsive hydrogels are theoretically capable of generating temporally complex delivery profiles such as pulsatile deliveries. In a study done by Hu et al, 43 the pulsatile release of vitamin B12 from a gelatin ferrogel was investigated. Ferrogels were exposed to5-minute high-frequency magnetic field (HFMF) pulses with 180 minutes of no magnetic field in between each stimulation. A burst increase in the release rate was observed during each stimulation period ( Figure 7). However, a gradual reduction in the release rate was observed over time.  In another study by Emi et al., 75 magnetically deformable alginate hydrogels were used to generate pulsatile mitoxantrone delivery profiles. However, in this study, pulsatile profiles were produced that were specifically similar to those demonstrated to be highly effective in killing melanoma cells in vitro (one 1-hr pulse of increased mitoxantrone per day for 3 days). This required generating pulsatile delivery profiles over days rather than hours. However, just as in the studies described above, 43,64 over the course of these 3-day experiments, the amount of drug released during magnetic stimulation significantly decreased each day, resulting in pulsatile delivery profiles with diminishing pulse heights. However, to compensate for this, Emi et al. used higher frequencies of magnetic stimulation on subsequent days to maintain delivery rates. This progressive increase in frequency resulted in pulsatile delivery profiles with uniform pulse heights ( Figure 9A). As mentioned above, generation of multi-therapeutic, sequential delivery profiles is also critical in directing biological processes pertinent to injury and disease. While little work has been done in using magnetically responsive hydrogels to achieve these types of complex delivery profiles, Kennedy Figure 9B). In another study by Tolouei et al., 76 a dual compartment biomaterial system was introduced that was composed of a gelatin outer compartment surrounding a 34 ferrogel inner compartment. This study went on to demonstrate that the outer compartment could initially release pro-inflammatory cytokines and the inner ferrogel could delay the delivery of anti-inflammatory cytokines (until magnetically triggered to do so). This 2-copartment biomaterial system was thus capable of generating sequences of pro-and anti-inflammatory cytokine deliveries using the timing of the magnetic stimulation to control the duration between these two deliveries.

Prospective strategies
Despite way where different compartments will be deformed when a magnetic is applied from different directions ( Figure 10B), then the directionality of drug release can be magnetically regulated. This compartmentalized approach can be applied to membrane reservoir system as described in Section 2 of this chapter as well to achieve control over multiple drug deliveries.    Figure 12, bubbles decorated with larger black dots, containing blue drug). Therefore, when stimulated using a relatively low-frequency AMF, the structures decorated with larger SPIONs will preferentially be disrupted, releasing their payloads (Figure 12, middle). When stimulated using a relatively highfrequency AMF, structures decorated with larger SPIONs will preferentially release their payloads (Figure 12, left).

Abstract
While inflammation can be problematic, it is nonetheless necessary for proper tissue regeneration. However, it remains unclear how the magnitude and duration of the inflammatory response impacts regenerative outcome. This is partially due to the difficulty in temporally regulating macrophage phenotype at wound sites. Here, a magnetically responsive biomaterial system potentially capable of temporally regulating macrophage phenotypes through sequential, on-demand cytokine deliveries is presented.
This material system is designed to (i) rapidly recruit proinflammatory macrophages It has been estimated that 1 to 2% of the population in developed countries will experience a chronic wound over their lifespan. [1] Occurrence of chronic wounds are particularly common in growing elderly populations and those who are suffering from diabetes and obesity. [2] While there are several phases in wound healing (i.e., coagulation, inflammation, proliferation, and remodeling), [3][4][5][6] chronic wounds are typically the result of prolonged and/or uncontrolled inflammation. [2,7] Despite this, inflammation is an indispensable step in the wound healing process and sets the stage for proper regeneration by staving off infection, clearing the wound site of debris, and recruiting cells to the wound that play critical roles in tissue remodeling and re-vascularization. [3,6,8,9] In fact, studies have shown that suppressing the inflammatory response actually hinders proper wound healing. [4,10] Macrophages play a key role in regulating the inflammatory response and in directing the transition to later stages of the wound healing process. [11][12][13][14][15][16][17] We and others believe that regulating the time at which macrophages transition from coordinating an inflammatory response (Figure 1.a, Phase 1 (red)) to coordinating later pro-healing stages of the wound healing process (Phase 2 (blue)) may be key to understanding the role of inflammation in wound healing and in developing improved treatment strategies. [18][19][20] For example, it is apparent that proper healing requires an inflammatory phase that eventually transitions into anti-inflammatory, pro-healing phases. [21,22] However, it remains unknown how the duration of this inflammatory phase (Tih) impacts or can be used to optimize wound healing outcome. Moreover, optimal durations are likely different for different wounds and for different patients. This motivates the need for biomaterials that enable flexible control over the duration of this inflammatory period, 52 as both an investigative and clinical tool.
Here, we propose a biomaterial system designed to deliver immunomodulatory cytokines in a manner that can potentially regulate the inflammatory period's duration in a flexible and on-demand manner. The inflammation phase can be initiated by establishing a population of pro-inflammatory M1 macrophages through the delivery of proteins that recruit macrophages and polarize them towards M1 phenotypes (Figure 1.b, M0 to M1): for example, Monocyte Chemoattractant Protein-1 (MCP-1), [23,24] and Interferon Gamma (IFN-γ). [25] Transition from inflammatory to healing phases requires establishing a population of anti-inflammatory M2 macrophages (e.g., alternatively activated M2a, Mb, and Mc phenotypes). This can be triggered through the delivery of other proteins at the wound site: for example, Interleukin-4 (IL-4), [26,27] and Interleukin-10 (IL-10), [26,27] (Figure 1.b, M0/M1 to M2). continue until, (iii) a magnetic gradient is applied that deforms the inner compartment, releasing anti-inflammatory cytokines, which would (iv) direct macrophages to take on pro-healing phenotypes. Such a material system could enable control over the inflammatory period's duration simply by applying a magnetic gradient (from simple hand-held magnets or electromagnets) at the time point at which one wishes inflammation to transition into an anti-inflammatory phase.

Fabrication and imaging of the biomaterial system
The outer compartment gelatin scaffolds used in these studies were purchased as 2 x 12 x 7 mm GelFoam™ sponge sheets (Pfizer, Groton, CT) and cut into hollow disks (2- The inner ferrogel compartments were made similarly to those described in Cezar et al. [28] Briefly, alginate was dissolved in MES buffer (100 mM MES and 500 mM NaCl at pH = 6.0) containing HOBT and AAD crosslinker and was cast with iron oxide particles and EDC (100 mg mL -1 ) between two Sigmacote-treated glass plates that were separated by 2-mm spacers. During casting (~ 1 hour), a magnet was placed against one glass plate as to pull the iron oxide particles towards one side of the gel, yielding a biphasic structure. Individual biphasic ferrogels were cut into 4 x 2 mm disks using a biopsy punch and then washed in 50 mL deionized water for 3 days (with water being exchanged twice a day) so that they would fully swell and become void of residual reagents. Ferrogels were then frozen at -20 ºC overnight and lyophilized. Lyophilized ferrogels were prepared for imaging by cross sectioning them using a sharp razor, sputtercoating in gold, and imaging as described above for the outer gelatin scaffolds.

Macrophage recruitment studies
In their culture flasks, RAW 264.7 mouse macrophages were rinsed in PBS, BioTek Gen5 software was used to quantify DAPI-nuclei count from these blue-channel images.

Magnetic stimulation of ferrogels
Ferrogels were magnetically stimulated using 0.5" x 0.5" x 0. clamps. This arrangement allowed ferrogel samples to be in close proximity to the magnetics when the magnets were raised (though the magnets did not physically touch the vials) and far enough away from the magnets (~10 cm) when the magnets were lowered, allowing the ferrogels to fully compress and conform back to their original uncompressed thickness between each cycle.

Cytokine time course release studies
Outer compartment gelatin scaffolds were unpacked and punched to shape in a lyophilized state. Thus, to load them with cytokine, concentrated solutions of protein were prepared and added dropwise directly to the dehydrated scaffolds. It was determined beforehand that when adding liquid to these scaffolds in this manner, they could fully absorb no more than 40 μL of solution. Thus, when loading the scaffolds, concentrated solutions were prepared such that the desired amount of protein to be loaded in the

58
Release studies from ferrogels followed a similar procedure. As described above, ferrogels were prepared with the final step being lyophilization, thus producing macroporous and dehydrated samples. Dried ferrogels were placed in scintillation vials with the Fe3O4-free region facing up. It was determined beforehand that when adding liquid to these ferrogels that they could fully absorb no more than 20 μL of solution.
They were therefore loaded using desired weights of protein dissolved in 20 μL of PBS (e.g., 1000 ng IL-4 in 20 μL PBS). Ferrogels were left to absorb the protein overnight in capped vials. Ferrogels were then rinsed in PBS with 1% BSA for 3 days to remove excess unincorporated protein, which reduced unstimulated baseline release. Ferrogels were then periodically sampled as described with the gelatin scaffolds, with sample media being fully removed and replaced with fresh media at each timepoint. Collected samples were quantitatively analyzed for IL-4 or IL-10 release using ELISA.

Statistical analyses
All quantitative data presented in this communication are represented as a mean ± standard deviation with 4 replicates (N = 4). Because only one-to-one statistical comparisons were made in this study (i.e., no multiple comparisons), student t-tests (twotailed distributions, heteroscedastic) were used to calculate p-values with p < 0.05 being our benchmark for significance (Microsoft Excel).

Results and discussion
This two-compartment biomaterial system comprises an outer gelatin scaffold and an inner biphasic ferrogel (Figure 2.a). The outer compartment exhibited an interconnected macroporous structure designed to permit rapid cell infiltration ( Figure   59 2b). Also, by virtue of being made from gelatin (a hydrolyzed form of collagen), this gelatin scaffold presents binding motifs for cell binding, motility, and spreading. [28,29] For the inner compartment, we utilized a biphasic ferrogel with an Fe3O4-laden region on the top half of the cylindrical gel and an Fe3O4-free, porous, and deformable region on the bottom (Figure 2.c). The particular ferrogel formulation adopted here (1 wt% alginate, 7 wt% Fe3O4, 2.5 mM adipic acid dihydrazide cross-linked, freeze-dried at -20ºC) was previously shown to be optimal in terms of providing magnetically triggered deliveries. [30,31] The outer porous gelatin scaffold was designed to provide initial deliveries of pro-  The inner compartment of this biomaterial system (Figure 2.c) was designed to provide delayed, on-demand, and magnetically triggered delivery of anti-inflammatory cytokines (e.g., IL-4 and IL-10). These biphasic ferrogels were designed so that cytokines could be loaded in their Fe3O4-free regions and released in earnest when magnetic gradients were used to compress the Fe3O4-free regions (Figure 4.a, white region of ferrogel compresses when a hand-held magnetic is subjacently applied). See MovieS2.mov in Supporting Information for a movie of a biphasic ferrogel being magnetically compressed repeatedly at 1.4 Hz. Cytokine release rates prior to magnetic stimulation were kept at low levels by thoroughly rinsing ferrogels, as to remove excess cytokines that were not well-incorporated. Additionally, cytokine retention prior to magnetic stimulation was likely aided by the use of alginate as the polymeric constituent of these ferrogels. Alginate is heparin-mimicking, and heparin is known to bind strongly to a wide variety of cell-secreted proteins. In these studies, magnetic gradients were applied over the course of 3 hours, but with different temporal profiles ( If this delayed IL-10 release was desired on day 5 rather than 3, magnetic stimulation could be applied on day 5 rather than day 3 (Figure 4.d, solid curve). This ability to control the time at which anti-inflammatories are earnestly released could provide a powerful tool for investigating how the duration of the inflammatory response impacts wound healing outcome. Magnetic stimulation can also potentially be used to repetitively deliver anti-inflammatory cytokines on subsequent days to prevent an inflammatory response from resurging. For example, when loaded with 1000 ng of IL-4, baseline levels of IL-4 were released prior to magnetic stimulation. But, release rates were dramatically enhanced when stimulated on day 4 using Stimulation Profile B (Figure 4.e, compare slope of curve before 96 hours to the slope from 96 to 99 hours). The rate of IL-4 release could be subsequently enhanced on days 5 and 6 when magnetically stimulated on those days (Figure 4.e, enhanced slopes at 120 and 144 hours). These magnetically stimulated release rates on days 4, 5, and 6 were significantly higher than control gels upon which no magnetic stimulation was applied (Figure 4.f). These studies demonstrate our ability to control the timing and rate of these anti-inflammatory cytokine deliveries in an ondemand, magnetically prescribed manner.
The described biomaterial system could improve control over the inflammatory response in wound healing applications by locally regulating macrophage phenotype through carefully timed immunomodulatory cytokine deliveries. There is a growing preponderance of evidence suggesting that regulating macrophage phenotype vs. time is critical to achieving desired outcomes in wound healing and regenerative therapies, [32][33][34][35][36] and that sequenced deliveries of immunomodulatory cytokines can provide a means for this temporal regulation. [26,37] In fact, previous studies have designed scaffolding materials to release pro-and anti-inflammatory cytokines at different rates in an attempt to temporally control macrophage phenotype. [19,[38][39][40][41] While these studies yielded promising results in their ability to influence macrophage phenotype in vivo, statistically significant improvements in regeneration were not observed (e.g., larger or more wellorganized vessels/tissues). This could have been due to the inability to explicitly alter and optimize the timing of different cytokine deliveries (i.e., having the delay time of antiinflammatory cytokines be a variable parameter between conditions). The biomaterial system described here could enable explicit control over the timing of these deliveries, without having to alter the chemistry or structure of the implantable scaffold material between experiments. It should be noted, however, that with this material system's current formulation, macrophages initially recruited to the outer compartment may be exposed to baseline levels of anti-inflammatory cytokines diffusing out of the inner ferrogel ( Figure.  concentration, polymer type, crosslinking density). Such a tuned biomaterial system will need to be tested in order to verify that this material system is capable of temporally regulating macrophage phenotype through magnetic stimulation.
In sum, we have developed a biomaterial system capable of initially delivering pro-inflammatory cytokines (MCP-1 and IFN-γ) from a macroporous gelatin structure capable of facilitating macrophage infiltration and growth. The amount of inflammatory cytokine release was dependent on the amount of cytokine loaded in the structure. This biomaterial system was also integrated with a biphasic ferrogel that was capable of 68 delivering anti-inflammatory cytokines (IL-4 and IL-10) in a delayed and magnetically triggered manner, using common hand-held magnets. The rate of magnetically stimulated delivery could be regulated by using different magnetic stimulation profiles and the timing of delivery could be regulated simply by choosing when to apply magnetic stimulation. This biomaterial system thus has the potential to enable experimental investigations into how the rate and timing of pro-and anti-inflammatory cytokine deliveries impact biological process critical in wound healing applications. Finally, this material system could also provide the material means to therapeutically implement optimized sequential cytokine deliveries, while retaining a high degree of clinical adaptability by enabling real-time alterations in delivery profiles.

Supporting Information: Appendix A
Additional experimental details and supplemental figures are provided in Supporting Information in Appendix A. 69 3.5

Abstract
Sequential protein release is required in regulating many biological processes that underlie injury and disease. A magnetically responsive dual-compartment biomaterial was therefore designed and successfully applied to provide on-demand sequential release of proteins relevant to specific therapies that would benefit from sequential release. The delayed enhancements in release rate where the timing (between days 1 and 8) and rate of release (0.2 to 1 ng/hr) were externally controlled through the temporal profile of magnet application and the frequency of that application. This biomaterial system can be used to investigate how the timing and sequence of protein deliveries impacts the biological processes that underlie cancer immunotherapy, tissue vascularization, and bone regeneration (and potentially in many other therapeutic areas) and can be used to experimentally optimize deliveries in these therapies.

Introduction
Hydrogels have been commonly used as biomaterials in drug delivery and tissue engineering applications due to their biocompatibility and versatility. 1-3 These drug delivery materials can potentially control important biological processes that need to be regulated to treat injury and disease. However, most biologies are sequential in nature and require sequential presentations of bio-instructive factors for proper regulation. For example, cancer is the second most common cause of death in the United States and accounts for nearly 1 of every 4 deaths. 4 This emphasizes the necessity of finding effective cancer treatment strategies. One promising cancer treatment strategy is biomaterials-based immunotherapy in which the immune system of the patient's own body is reprogramed in order to initiate an immunological attack against cancer cells. [5][6][7] In this approach, first, dendritic cells (DCs) need to be recruited to the biomaterial by releasing a DC recruitment factor (Factor I) (Fig 1.A.i, ii). Once a large number of DCs are resident in the biomaterial (Fig 1.A.iii), they can become activated when presented with a cancer antigen (Factor II) (Fig 1.A.iv). Activated DCs would then migrate out of the biomaterial towards lymph node (Fig 1.A.v), triggering an immunological attack against cancer. Therefore, sequential release of recruitment factor followed by release of activating factor could potentially improve control over regulating the biological processes pertinent to biomaterial-based cancer immunotherapies.
Beyond cancer treatments, regulation of vascular growth can be of great potential in the treatment of different cardiovascular diseases (CVDs), which are the most common cause of death in the United States and worldwide. 8 It has been reported that CVD was the main cause of more than 50% of deaths in 2010. 9 Additionally, regeneration of tissues after surgery or injury often involves regulating the growth of new vascular networks. [10][11][12] Pericyte cells play a key role in vessel formation and presentation of several growth factors can regulate this process. 13 For example, growth of new blood vessels can be initiated by an initial presentation of angiogenic factors (Factor I) which instructs pericyte cells to detach from the endothelium of nearby vasculature. This detachment destabilizes the endothelium and allows small vascular sprouts to grow away from the existing blood vessel (Fig 1.B.i, ii). These nascent sprouts are thin, unorganized, and not mature enough to efficiently perfuse blood through them (Fig 1.B.iii). Hence, an additional maturation factor (Factor II) is subsequently released to recruit pericyte cells back to neovessels (Fig   1.B.iv), which in turn helps neovessels mature into a thicker and more interconnected network (Fig 1.B.v). Finally, sequential delivery of bio-instructive factors can be of potential value in regenerating bone tissues. Each year more than 6 million bone fractures occur in the United States, leading to approximately 900,000 patient hospitalizations. 14 Biomaterial scaffolds can be promising substitutes for traditional autogenic and allogenic grafting since they can decrease the problems associated with donor site sensitivity, morbidity, and limited availability of these grafts. 15,16 Bone regeneration also is naturally regulated by a sequence of growth factor presentations. 17 First, osteoprogenitor cells need to be recruited to the scaffold by releasing a bone progenitor recruitment factor (Factor I) (Fig   1.C.i, ii). After establishing a population of these progenitor cells in the biomaterial (Fig 1.C.iii), they can be differentiated down the osteogenic lineage by exposing them to a osteo-differentiation factor (Factor II) (Fig 1.C.iv). Differentiated bone cells would then start secreting their own robust bone matrix, which is a vital step in regenerating new bone tissues (Fig 1.C.v).Therefore, in order to better regulate these regenerative processes, sequential delivery of bone progenitor recruitment and differentiation factors is necessary.
In previous studies, hydrogels were demonstrated to have sequentially protein release capabilities using formulations containing phases with different degradation rates.
However, the timing between these two deliveries was not capable of being regulated after implantation or injection of these biomaterials. [18][19][20] Additionally, these delivery profiles can more aptly be described as dual deliveries with different rates and not sequential release per se (i.e., on burst release followed by a second, delayed burst release). The biomaterial system presented here was specifically designed to provide sequential delivery profiles where there is an initial burst release of one factor followed by a magnetically triggered, delayed release of a second factor. This was achieved by composing the biomaterial with two compartments (Fig 2.A.i, ii). Compartment 1, initially releases Factor I and has a functionality to maintain recruited cells (Fig 2.A.iii, iv). Compartment 2 is capable of releasing Factor II in an on-demand manner when remotely stimulated with a magnetic field (Fig 2.A.v). This study aimed to demonstrate the ability to generate these sequential delivery profiles for specific recruitment factors

Fabrication and characterization of biomaterial system
The outer compartments of these two-compartment biomaterial systems were Emission-SEM and Energy-dispersive X-ray Spectroscopy (EDS) for elemental mapping.
The inner compartment ferrogels were made of alginate according in a similar manner to the biphasic ferrogels described elsewhere. [21][22][23] Briefly, alginate (at 1%wt) was dissolved in MES buffer (100 mM MES and 500 mM NaCl at pH = 6.0) with AAD and HOBt. This mixture was then mixed with iron Oxide particles (Fe3O4 < 5 μm) and 100 mg/mL EDC. Next, the mixture was cast between two sigmacote-treated glass plates separated by 2 mm spacers. During casting (~ 30 minutes), a magnet was placed on top of the glass plate to pull the iron oxides to the top of the ferrogel, resulting in a biphasic design. Ferrogels were then cut into 4 x 2 mm (diameter-thickness) disks using 4-mm biopsy punches. Gels were then washed in deionized water for 3 days with water being changed 2 to 3 times a day, this was done in order to remove residual reagents and let them swell. Next, to achieve a porous structure, gels were frozen overnight at -20 °C and lyophilized. For SEM imaging, ferrogels were cut with a sharp razor, exposing their cross section and prepped for SEM imaging following the same protocol as described above for gelatin outer compartments.

Magnetic stimulation of ferrogels
Two different set up were used to magnetically stimulate the Ferrogels, both involving the use of 0.5"x0. pistons. Thus, when the piston was in the up position, a ferrogel would be exposed to a strong magnetic field (measured to be 5 kGauss). When the piston was in the down position, the magnetic field was weak (measured to be < 10 Gauss). Thus, the speed of the motor dictated the magnetic stimulation frequency. It was determined that this apparatus could magnetically stimulate ferrogels at frequencies up to 14 Hz.

Protein release studies
In order to load proteins to these gels, concentrated solutions of proteins in PBS were made. It was determined that each outer gelatin compartment had the capacity to absorb 40 ml of solution which was used as the basis to calculate protein loading concentrations (i.e., loading of 1000 ng protein would require preparation of a solution containing 1000 ng GM-CSF in 40 ml PBS). Sigmacote-treated scintillation vials (to prevent protein adsorption to the surface of the vials) were used as containers for these gels. Protein carrying solutions were added dropwise to these outer compartments for 86 loading. Vials were capped, and gels were left at room temperature overnight for full absorption of the protein into the gels. Release studies began the next day when gels were submerged in PBS with 1% BSA (t = 0). 1 ml samples were collected periodically and replaced with fresh media each time. Collected samples were stored in 1.5 ml lowadsorption tubes in the freezer. After collecting all samples, samples were thawed and quantified for protein content (i.e., GM-CSF, VEGF, SDF-1α) using ELISA.
Inner compartment fabrication resulted in lyophilized alginate biphasic gels with a layer of iron-oxide-free porous alginate on one side of the gel and an iron-oxide saturated layer with smaller pores on the other side of the gel. The loading of these ferrogels and release studies from them were similar to those described for the outer gelatin compartments above. However, the absorption capacity of these ferrogels were 20 ml.
Ferrogels were placed in scintillation vials with their iron-oxide free region facing up.
Next, a 20 ml solution containing protein was added to them dropwise. Vials were capped and left in order for gels to absorb the proteins overnight. Ferrogels were flipped over so that their iron-oxide-saturated regions faced upwards (so that a magnet applied under the vial would deform the ferrogel in a downward motion) and then rinsed in PBS with 1% BSA for between 1 to 4 days to remove proteins that were not well integrated in the gel structure. Samples were then taken at different times and fresh media was replaced at each time point. During these time course release experiments, ferrogels were magnetically stimulated at a number of time points and at various frequencies, depending on the experiment. ELISA was performed on the collected samples to quantify the concentration of the released proteins from ferrogels (HSP27, PDGF, BMP2).

Data representation and statistical analysis
Analysis of variance (ANOVA) with post-hoc Tukey HSD (Honestly Significant Difference) test was performed in order to determine statistically significant differences when multiple conditions and comparison were made. The numerical values presented in the graphs were represent means ± standard deviations from 4 independent replicates (N = 4). P-values less than 0.05 were considered significant.

Characterization of the two-compartment biomaterial system
The two-compartment biomaterial system was made of an outer gelatin compartment and an inner biphasic ferrogel nested within the outer gelatin compartment (Fig 3. A). The gelatin outer compartment was highly porous (Figure 3. B), which would be desirable for allowing fast diffusive release of load proteins (due to high surface area) and potentially efficient penetration of recruited cells. Additionally, by virtue of being made from gelatin, this outer compartment contained cell-binding integrins needed to have recruited cells attach the scaffold and proliferate within the scaffold. The inner biphasic ferrogel compartment contained a Fe3O4 saturated region on the one side of the gel and a highly porous Fe3O4-free region on the opposite side of the gel (Fig 3. C. i).
This biphasic structure was capable of deforming in the presence of magnetic field (Fig 3. C. ii).

Release characteristics of the outer gelatin scaffold
The outer compartment's porous gelatin scaffold was capable of initially releasing proteins (proteins described herein as "Factor I") in a rapid manner. Specifically, DC recruitment factor (GM-CSF) released mostly within the first 24 hours (Figure 4.

Release characteristics of the inner ferrogel
The inner compartment provides magnetically triggered, delayed, and on-demand release of proteins that can be used to direct the behavior of (i.e., program) cells recruited to the outer compartment. For example, a model cancer antigen's (HSP27's) release rate drastically increase when ferrogels inner compartments are magnetically stimulated on days 4 ( Figure 5. A, slope of blue curve increases in the blue shaded region of the curve) or on day 7 (slope of red curve increases in the red shaded region), depending on when the magnetic stimulation is applied (i.e., on day 4 or 7, respectively). Note that the magnetic stimulation used in these studies was at constant 1.4 Hz for 4 hours using the rocker. These on-demand, delayed enhancements in release rate could be used to optimize the timepoint at which recruited DCs are earnestly presented with cancer antigen for biomaterials-based cancer immunotherapy applications.

Strategies for magnetically controlling the rate of delayed release
While the delayed release capabilities outlined above may be of use in improving the timing of deliveries in cancer immunotherapies, re-vascularization therapies, and bone regeneration, the rate of release when magnetically stimulated is also a critical parameter for optimization. Here, strategies were explored for using alterations in the magnetic stimulation profile to regulate the release rate during magnetic stimulation. A previous study demonstrated that periodically turning on and off sinusoidal magnetic stimulations could actually improve triggered release rates compared to continuous sinusoidal magnetic stimulation. 24 Here, this principle was investigated further by adjusting parameters associated with pulsing the magnetic stimulation regiment. Namely, ferrogel inner compartments were exposed to pulsed magnetic stimulation regiments where the frequency and duration of magnetic stimulation pulses were changed vs. time.
It was demonstrated that applying different pulsed magnetic stimulation regiments could be used to control the rate of protein release during magnetic stimulation. For 92 example, after 3 days of diffusive release, ferrogels containing HSP27 were magnetically stimulated using one of two different pulsed regiments: either a regiment switching between (i) 10 minutes of magnetic stimulation at 5 Hz followed by 50 minutes at 1 Hz, repeated over 4 hours (Figure 6.

Discussion
These two-compartment biomaterial systems may provide improvements in a wide range of biomedical applications, including biomaterial-based cancer immunotherapies, therapies involving regeneration of vascular networks, and in bone regeneration. For example, the sequential deliveries of DC recruitment factors followed  have been due to the specific temporal profiles of VEGF and PDGF delivery from the system. The differential degradation approach adopted by Richardson et al. yielded VEGF and PDGF release profiles that were more of different rates as opposed to sequential per se. That is, there was not a drastic (albeit slight) increase in PDGF release at later time points (i.e., dramatically delayed PDGF increases in release rate, as observed here in using magnetically responsive ferrogels). This lack of sequential delivery may have not property coordinated the sequence of pericyte detachment, vascular sprouting, vascular invasion, pericyte re-recruitment and attachment, and sprout maturation required to generate mature vasculature. The delivery profiles achieved by the two-compartment biomaterial system described here may more properly coordinate these sequential biological events. Furthermore, the on-demand capabilities of this two-compartment system may enable critical optimization in the timing allowed for sprouting and sprout infiltration prior to initiating vascular maturation.

97
Finally, the two-compartment magnetically responsive biomaterial system described here might be beneficial for optimizing deliveries in treating bone defects, injuries, and diseases. Regenerating bone requires coordinated sequence recruitment, proliferation, and differentiation events, which can each potentially be coordinated through protein deliveries. In an attempt to coordinate these events, Lee et al. 28 developed a biomaterial that contained two factors with the goal of enhancing the number of calcium phosphate matrix-producing osteoblasts: TGFβ (to enhance the population of bone progenitors resident in the regenerative scaffold) and BMP2 (to differentiate those bone progenitors into osteoblasts). While this strategy demonstrated that BMP2-loaded scaffolds yielded significantly higher amounts of bone-matrix than controls, dual-loaded BMP2/TGFβ scaffolds did not produce more bone matrix than only BMP2-loaded scaffolds (and may in fact have yielded less bone matrix). Again, this result may have been due to the timing of how and when these proteins were presented to bone progenitors as they entered the scaffold from surrounding tissues. For example, while TGFβ is known to enhance cell proliferation, it is also known that differentiated cells do not proliferate well. 28  signaling), it may be possible to generate a more robust population of bone-matrix-98 producing osteoblasts. The dual-compartment material system presented here may afford coordination of this sequence and furthermore be capable of optimizing the timing of this sequence for maximized bone matrix production.

Conclusions
In this study, a biomaterial system was developed and its ability to sequentially release two different proteins pertinent to three therapies was demonstrated. This system was capable of rapidly releasing initial factors (GM-CSF, VEGF, or SDF-1a) from a porous gelatin outer compartment that was designed to facilitate cell infiltration. The amount of this delivery depended on the amount of the factor that was loaded into the outer compartment. Additionally, this biomaterial system contained a magnetically responsive ferrogel inner compartment, which was able to produce enhanced delivery of its payload (HSP27, PDGF, or BMP2) at a time dictated by an externally applied magnetic signal. It was also demonstrated that the rates of these delayed and magnetically triggered protein deliveries could potential be regulated by altering the frequencies and/or temporal profile of the magnetic stimulation itself.

4.7
Summary and Prospective Research

Primary goals
The research described in this dissertation was to investigate magnetically responsive drug delivery systems and develop a biomaterial system capable of controlling the rate and timing of different biomolecular deliveries in response to a remotely applied magnetic fields. More specifically, this doctoral research aimed to (1) develop a dual compartment biomaterial system capable of generating sequences of biomolecular deliveries in response to low-frequency, spatially graded magnetic fields, and (2) to investigate the applications of this developed biomaterial for delivering critical biomolecules in several therapeutic applications that may be enhanced through sequential deliveries.

Summary of individual chapters
While Chapter 1 broadly motivated the work that is contained in this dissertation, Chapter 2 elaborated on this motivation by discussing existing hydrogel-based magnetically responsive biomaterials with some highlighted examples and in terms of mechanisms of magnetically triggered release and parameters that influence release characteristics. A few case studies were examined where magnetically responsive 105 hydrogels were used to generate temporally complex and multi-drug delivery profiles.
However, Chapter 2 in fact underscored how not many magnetically responsive biomaterials had been introduced that demonstrated the capability of regulating temporally complex, multi-drug deliveries. This underscores the urgency for novel biomaterial systems with these capabilities. Thus, in Chapters 3 and 4, a novel biomaterial system was described that was specifically designed to generate temporally complex, multi-drug delivery profiles in response to remotely applied magnetic stimuli.
Chapter 3 focused on developing and demonstrating novel delivery capabilities for a biomaterial system possibly capable of regulating the inflammation response in wound healing applications. This two-compartment biomaterial system was designed to initially release factors that could recruit a population of pro-inflammatory macrophages (i.e., by delivering MCP-1 and IFNg from an outer compartment that was porous and presented integrins for macrophage binding). Magnetically triggered release of antiinflammatory factors (i.e., IL4 and/or IL10 from a magnetically deformable ferrogel as the inner compartment) was designed to switch off the inflammation and the promote healing processes.
Chapter 4 explored the use of this two-compartment biomaterial system in additional applications where remote regulation of the timing of sequential biomolecular deliveries may be of high clinical value. Specifically, the dual compartment biomaterial system was loaded with biomolecules pertinent to cancer immunotherapy, tissue vascularization, and bone regeneration. Biological processes relevant to these three applications can each benefit sequential regulation of biosocial events through sequenced biomolecular deliveries. For instance, in biomaterial-based cancer immunotherapy 106 applications, a goal for potentially improving these therapies is to increase the number of the activated dendritic cells. This possibly can be done by releasing DC recruitment factor while holding on to the activating factor inside the ferrogel and have it be released to recruited DCs only when the DC population is relatively high. This would lead to increased numbers of activated immune cells and a stronger anti-cancer immune response. Indeed, the two-compartment biomaterial system developed was demonstrated to sequentially deliver DC recruitment factors (i.e., GM-CSF) followed by magnetically triggered delivery of DC activation factors (i.e., single stranded DNA and a model protein antigen, HSP-27). In tissue vascularization applications, the aim was to demonstrate sequential deliveries that could direct the sprouting of immature vascular sprouts, followed by coordination of events that lead to maturation of those vascular sprouts.
Indeed, the two-compartment biomaterial system was demonstrated to sequentially deliver pro-angiogenic sprouting factors (i.e., VEGF) followed by magnetically triggered pro-maturation factors (i.e., PDGF). In bone regeneration applications, the aim was to demonstrate sequential deliveries that could first recruit bone progenitor cells followed by differentiation of those recruited progenitor cells down the osteogenic lineage. Again, this work successfully demonstrated the ability to first release a bone progenitor recruitment factor (SDF-1a) followed by magnetically triggered release of an osteodifferentiation factor (BMP-2).

Impact and importance of this work
The biomaterial system developed and demonstrate in this dissertation can potentially be used to help scientists better understand how the dosing, sequence, and timing of the drug release can impact the outcome in wound healing, cancer 107 immunotherapies, and tissue engineering (i.e., neo-vascularization of and establishing populations of tissue-specific cell types within defect sites). This can help scientists optimize different drug delivery regiments before moving on to clinical trials and implementing the dosing on patients. Also, the on-demand nature of these magnetically responsive materials will help enable rapid optimizations since the material itself does not need to be altered between experiments (i.e., the same material system is capable of producing a wide variety of delivery profiles whereas a traditional biomaterial must be reformulated to produce different delivery profiles). Furthermore, the on-demand nature of these magnetically responsive biomaterials would enable real-time alterations in delivery schedules, which would provide a high degree of flexibility for clinicians to alter the course of therapy in real time according to updates in patient prognosis. Finally, from a practical standpoint, the magnetic stimulations needed for these triggered deliveries involve benign fields from simple, inexpensive, hand-held magnets.
Another significant aspect of this project involves the fact that this developed drug delivery system can be made with variety of biomaterials in different shapes and sizes and can be loaded with many different bioactive molecules, depending on the desired application. For example, the porosity of the outer compartment can be adjusted for controlling the diffusion rate of the drug as well as the chemistry of the polymer which can be easily modified to attach different molecules to it. Also, these biomaterials can be designed to degrade in body within a desired timeframe, which holds up the need to take out the biomaterial once it is no longer required. This is particularly desirable in tissue engineering applications where the body will replace the scaffold with its own native extracellular matrix.
in outer section for recruiting MSCs and bone morphogenetic protein-2 (BMP-2) loaded inner ferrogel can be placed on top of the cells. After stem cell recruitment to the biomaterial, stimulated release of BMP-2 from the ferrogel will differentiate the MSCs to bone lineage. For counting the recruited cells, we can fix and stain the gel holding the cells with DAPI for cell nuclei and Phalloidin to stain the F-actin and images were taken using confocal microscope. For osteo-differentiation, the culture media can be collected on daily basis and analyzed using ELISA for "osteocalcin" secretion as indicator of early stage osteo-differentiation and for "Alizarin Red" to stain calcium phosphate as an indicator of late stage osteo-differentiation.   n.s.